Cyclodextrin polymer compositions for use as drug carriers

ABSTRACT

This invention discloses compositions of cyclodextrin polymers for carrying drugs and other active agents. Compositions are also disclosed of cyclodextrin polymer carriers that release drugs under controlled conditions. The invention also discloses compositions of cyclodextrin polymer carriers that are coupled to biorecognition molecules for targeting the delivery of drugs to their site of action.  
     The advantages of the water-soluble cyclodextrin polymer carrier are:  
     (1) Drugs can be used based on efficacy without solubility or conjugation requirements.  
     (2) Drugs can be delivered as macromolecules and released within the cell.  
     (3) Drugs can be targeted by coupling the carrier to biorecognition molecules.  
     (4) Synthesis methods are independent of the drug to facilitate multiple drug therapies.

RELATED PATENT APPLICATIONS

[0001] This is a continuation-in-part of PCT/US99/30820, filed Dec. 27,1999, which is a continuation-in-part of U.S. patent application Ser.No. 09/223,055, filed Dec. 30, 1998, now U.S. Pat. No. 6,048,736, issuedApr. 11, 2000. The contents of those applications are incorporatedherein.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention discloses methods for preparing compositions ofcyclodextrin polymers for carrying drugs and other active agents fortherapeutic, medical or other uses. Methods are also disclosed forpreparing cyclodextrin polymer carriers that release drugs and otheractive agents under controlled conditions. The invention also disclosesmethods for preparing compositions of cyclodextrin polymer carriers thatare coupled to biorecognition molecules for targeting the delivery ofdrugs and other active agents to their site of action.

DESCRIPTION OF THE PRIOR ART

[0003] The pharmacokinetics of many anti-viral and anti-cancer drugs andother active agents that penetrate cells are not easily controlled.Therefore, there is a need for carriers of drugs that facilitate theirsolubility, delivery and effectiveness. When drugs are bound to polymersof the prior art they can be taken up at the cell surface andtransferred to the nucleus. This permits modulation of drug uptakethrough cell surface properties. Also, drug release can be controlledusing specific enzymes and other conditions within the cell.

[0004] Drugs and other active agents delivered as macromolecules throughpolymer carriers have gained acceptance as a way for improving nucleicacid therapies. Also, the prior art now employs drug-polypeptidecomplexes to re-direct drugs to selected target cells. However, becausecyclodextrin polymers of the prior art lack any specific releasemechanism, many of the advantages of cyclodextrins are limited.

[0005] The prior art of cyclodextrins has disclosed their use inlabeling materials for in vitro testing (Kosak, PCT WO 91/05605, 1991),and in drug preparations (fitai, et al, U.S. Pat. No. 4,523,031 and4,523,037).

[0006] The preparation and use of individual cyclodextrins conjugated tobiorecognition molecules as drug carriers is disclosed by Weinshenker,U.S. Pat. No. 5,068,227; 1991, where each coupling site is limited toone drug molecule. However, Weinshenker makes no disclosures orsuggestions for any cyclodextrin polymers and they cannot be made withthe synthesis methods disclosed.

[0007] Review articles on the pharmaceutical applications ofcyclodextrins have identified many problems due to the high turnoverrate between inclusion complex formation and dissociation. Stella, V.J., et al., Pharmaceut. Res. 14, 556-567 (1997), report that even withthe strongest theoretical binding constants, as soon as the complex offree cyclodextrin and drug is diluted in the bloodstream, over 30% iscalculated to dissociate. Also, Rajewski, R., et al, J. Pharm. Sci. 85,1142-1169 (1996), solubilized the anti-cancer drug Taxol withcyclodextrins. They reported on page 1145 that “any attempt to dilutethe samples resulted in erratic precipitation” due to competitivedisplacement factors found in plasma. Because of these problems,cyclodextrins in the prior art are used for solubilizing and stabilizingcertain drugs before or during administration but are not suitable forcarrying and delivering drugs in the bloodstream.

[0008] The cyclodextrin polymer carriers of the instant inventionovercome these problems and provide the new function of controlledrelease of drugs, which is not disclosed or suggested by the prior art.

SUMMARY DISCLOSURE OF THE INVENTION

[0009] It has been discovered that the water-soluble (or colloidal)cyclodextrin polymer carriers of the instant invention overcome theproblems with individual (monomeric) cyclodextrins in the prior art. Theinstant invention provides new properties and unexpected advantages. Inits simplest form, a cyclodextrin polymer carrier comprises acyclodextrin polymer that has a nucleic acid or other active agentcompletely entrapped within it.

[0010] In one embodiment, the water-soluble (or colloidal) cyclodextrinpolymers of the instant invention overcome the problem of low carryingcapacity of individual cyclodextrins. Also, by complete entrapment ofthe guest molecules, the problem of losing drug or other active agent bydiffusion when diluted in vivo, is solved. In another embodiment, theinvention also provides a means for controlled release of the entrappeddrug in vivo, which was not possible in the prior art of cyclodextrins.

[0011] In another embodiment, the invention also provides a means fortargeting the cyclodextrin polymer carrier by coupling it to abiorecognition molecule.

[0012] The advantages of the water-soluble cyclodextrin polymer carrierare:

[0013] (1) Drugs can be used that are designed for efficacy withoutsolubility or conjugation requirements.

[0014] (2) Drugs can be delivered as macromolecules and released withinthe cell.

[0015] (3) Drugs can be targeted by coupling the carrier tobiorecognition molecules.

[0016] These are new advantages and functions provided for drug carriertechnology that will also be useful for other drug deliveryapplications. These compositions and methods are unanticipated orsuggested in the prior art.

INDUSTRIAL APPLICABILITY

[0017] These CD polymer carriers can be used in many fields of medicineto deliver therapeutic drugs and other active agents through a varietyof routes including orally, nasally and parenterally. Other routesinclude various applications for delivery through ocular membranes andmucosal membranes, including the use of electric charge as iniontophoresis.

DESCRIPTION OF THE BEST MODES FOR CARRYING OUT THE INVENTION

[0018] For the purposes of disclosing this invention, certain words,phrases and terms used herein are defined as follows:

[0019] Active Agents.

[0020] Active agents function as the preferred guest molecules of theinstant invention. Active agents that are preferred in the instantinvention are chemicals and other substances that can form an inclusioncomplex with a cyclodextrin or cyclodextrin polymer and are inhibitory,antimetabolic, therapeutic or preventive toward any disease (i.e.cancer, syphilis, gonorrhea, influenza and heart disease) or inhibitoryor toxic toward any disease causing agent. Preferred active agents areany therapeutic drugs categorized in The Merck Index, Eleventh Ed.,Merck & Co. Inc., Rahway N.J. (1989) and those listed by Cserhati, T.,Anal.Biochem. 225(2), 328-332 (1995).

[0021] Active agents include but are not limited to therapeutic drugsthat include prodrugs, anticancer drugs, antineoplastic drugs,antifungal drugs, antibacterial drugs, antiviral drugs, cardiac drugs,neurological drugs, and drugs of abuse; alkaloids (i.e. camptothecins),antibiotics, bioactive peptides, steroids, steroid hormones, polypeptidehormones, interferons, interleukins, narcotics, nucleic acids includingantisense oligonucleotides, pesticides and prostaglandins.

[0022] Active agents also include any toxins including aflatoxins,ricins, bungarotoxins, iotecan, ganciclovir, furosemide, indomethacin,chlorpromazine, methotrexate, cevine derivatives and analogs includingcevadines, desatrines, and veratridine, among others. Also included butare not limited to, are;

[0023] various flavone derivatives and analogs includingdihydroxyflavones (chrysins), trihydroxyflavones (apigenins),pentahydroxyflavones (morins), hexahydroxyflavones (myricetins),flavyliums, quercetins, fisetins;

[0024] various antibiotics including derivatives and analogs such aspenicillin derivatives (i.e. ampicillin), anthracyclines (i.e.doxorubicin, daunorubicin, mitoxantrone), butoconazole, camptothecin,chalcomycin, chartreusin, chrysomicins (V and M), chloramphenicol,chlorotetracyclines, clomocyclines, cyclosporins, ellipticines,filipins, fungichromins, griseofulvin, griseoviridin, guamecyclines,macrolides (i.e. amphotericins, chlorothricin), methicillins, nystatins,chrymutasins, elsamicin, gilvocarin, ravidomycin, lankacidin-groupantibiotics (i.e. lankamycin), mitomycin, teramycins, tetracyclines;

[0025] various anti-microbials including reserpine, spironolactone,sulfacetarnide sodium, sulphonamide, thiamphenicols, thiolutins;

[0026] various purine and pyrmidine derivatives and analogs including5′-fluorouracil, 5′-fluoro-2′-deoxyuridine, and allopurinol;

[0027] various photosensitizer substances, especially those used forsinglet and triplet oxygen formation useful for photodynamic therapy(van Lier, J. E. In “Photodynamic Therapy of Neoplastic Disease”; KesselD., Ed., CRC Press, Boca Raton, Fla., 1990, Vol. I), includingmeso-chlorin e₆ monoethylenediamine (Mce₆), phytalocyanine, porphyrinsand their derivatives and analogs;

[0028] various steroidal compounds such as cortisones, estradiols,hydrocortisone, testosterones, prednisolones, progesterones,dexamethasones, beclomethasones and other methasone derivatives, othersteroid derivatives and analogs including cholesterols, digitoxins,digoxins, digoxigenins;

[0029] various coumarin derivatives and analogs includingdihydroxycoumarins (esculetins), dicumarols, chrysarobins, chrysophanicacids, emodins, secalonic acids;

[0030] various dopas, derivatives and analogs including dopas,dopamines, epinephrines, and norepinephrines (arterenols);

[0031] various antineoplastic agents or cell growth inhibitors such ascisplatins and taxanes including paclitaxel and docetaxel;

[0032] various barbiturates including phenobarbitone, amobarbital,allobarbital, pentobarbital and other barbital derivatives;

[0033] various benzene derivatives including amino-benzoic acid,bromobenzoic acid, benzocaine, benzodiazepines, benzothiazide,butyl-p-aminobenzoate;

[0034] various polypeptide derivatives;

[0035] various carboxylic acid derivatives such as bromoisovalerylurea,phenyl-butyric acid, phenyl-valeric acid;

[0036] Other active agents include, but are not limited to, diphenylhydantoin, adiphenine, anethole, aspirin, azopropazone, bencyclane,chloralhydrate, chlorambucil, chlorpromazine, chlorogenin, cinnamicacid, clofibrate, coenzyme A, cyclohexyl anthranilate, diazepam,flufenamic acid, fluocinolone acetonide, flurbiprofen, guaiazulene,ibuprofen, indican, indomethacin, iodine, ketoprofen, mefanamic acid,menadione, metronidazole, nitrazepam, phenytoin, propylparaben,proscillaridin, quinolone, thalidomide, thiamine dilaurylsulphate,thiopental, triamcinolone, vitamins A, D3, E, K3, and warfarin.

[0037] Other specific active agents are anti-viral drugs, nucleic acidsand other anti-viral substances including those against any DNA and RNAviruses, AIDS, HIV and hepatitis viruses, adenoviruses, alphaviruses,arenaviruses, coronaviruses, flaviviruses, herpesviruses, myxoviruses,oncornaviruses, papovaviruses, paramyxoviruses, parvoviruses,picornaviruses, poxviruses, reoviruses, rhabdoviruses, rhinoviruses,togaviruses and viriods; any anti-bacterial drugs, nucleic acids andother anti-bacterial substances including those against gram-negativeand gram-positive bacteria, acinetobacter, achromobacter, bacteroides,clostridium, chlamydia, enterobacteria, haemophilus, lactobacillus,neisseria, staphyloccus, and streptoccocus; any anti-fungal drugs,nucleic acids and other anti-fungal substances including those againstaspergillus, candida, coccidiodes, mycoses, phycomycetes, and yeasts;any drugs, nucleic acids and other substances against mycoplasma andrickettsia; any anti-protozoan drugs, nucleic acids and othersubstances; any anti-parasitic drugs, nucleic acids and othersubstances; any drugs, nucleic acids and other substances against heartdiseases, tumors, and virus infected cells, among others.

[0038] Nucleic Acid Active Agents.

[0039] For the purposes of this invention, certain nucleic acids arepreferred as a specific class of active agents directed against viraland other microbial diseases, against cancers, autoimmune and geneticdiseases. Specific nucleic acid active agents include anyanti-bacterial, anti-cancer, anti-fungal, anti-viral, anti-parasitic andanti-protozoan nucleic acids. They also include specific DNA sequencesused for gene therapy.

[0040] Nucleic add active agents include all types of RNA (includingmessenger RNA), all types of DNA, and oligonucleotides including probesand primers used in the polymerase chain reaction (PCR), hybridizationsor DNA sequencing. Also preferred are phosphodiester sense or antisenseoligonudeotides, sense or antisense oligodeoxynucleotides (ODN) and anysense or antisense oligonucleotides or oligodeoxynucleotides where thesugar-phosphate “backbone” has been derivatized or replaced with“backbone analogues” or linkages such as with phosphorothioates,phosphorodithioates, phosphoroamidates, alkyl phosphotriesters, ormethylphosphonates. Also preferred are any sense or antisenseoligonudeotides or oligodeoxynucleotides with non-phosphorous backboneanalogues or linkages such as sulfamates, 3′-thioformacetals,methylene(methylimino), 3′-N-carbamates, or morpholino carbamates.

[0041] Some preferred examples of synthetic oligonucleotides and ODNsare disclosed by J. F. Milligan, et al., J. Medicinal Chem. 36(14):1923-1937 (1993) and Y. Shoji et al., Antimicrob. Agents Chemotherapy,40(7):1670-1675 (1996). Also included are synthetic nucleic acidpolymers and peptide nucleic acids (PNA) disclosed by Egholm, et al,Nature 365:566-568(1993) and references therein, including PNA damps(Nucleic Acids Res. 23:217(1995)). Also included are nucleotide mimicsor co-oligomers like phosphoric add ester nucleic adds (PHONA),disclosed by Peyman, et al., Angew. Chem. Int. Ed. Engl. 36:2809-2812(1997). Also included are DNA and/or RNA fragments, and derivatives fromany tissue, cells, nuclei, chromosomes, cytoplasm, mitochondria,ribosomes, and other cellular sources.

[0042] Biocleavable Linkage or Bond.

[0043] For the instant invention, biocleavable linkages are defined astypes of specific chemical moieties or groups used within the chemicalsubstances that covalently or non-covalently couple and cross-link thecyclodextrin polymer carriers. They are contained in certain embodimentsof the instant invention that provide the function of controlled releaseof an entrapped drug or other active agent. Biocleavable linkages orbonds are defined here under distinct categories or types.

[0044] One category comprises the disulfide linkages and ester bondsthat are well known for covalently coupling drugs to polymers. For drugdelivery, they may be more useful for shorter periods in vivo since theyare cleaved in the bloodstream relatively easily. Ester bonds includethose between any acid and alcohol, and imidoesters formed from alkylimidates.

[0045] Another category comprises linkages or bonds that are morespecifically cleaved after entering the cell (intracellular cleavage).The preferred linkages for release of drugs within the cell arecleavable in acidic conditions like those found in lysosomes. Oneexample is an acid-sensitive (or acid-labile) hydrazone linkage asdescribed by Greenfield, et al, Cancer Res. 50, 6600-6607 (1990), andreferences therein. Also preferred are certain natural or syntheticpolypeptide linkages that contain certain amino acid sequences (i.e.usually hydrophobic) that are cleaved by specific enzymes such ascathepsins, found primarily inside the cell. Using the convention ofstarting with the amino or “N” terminus on the left and the carboxyl or“C” terminus on the right, some examples are: any polypeptide linkagesthat contain the sequence Phe-Leu, Leu-Phe or Phe-Phe, such asGly-Phe-Leu, Gly-Leu-Phe, Gly-Phe-Leu-Gly, Gly-Phe-Leu-Phe-Gly andGly-Phe-Phe-Gly, and others that have either of the Gly residuessubstituted for one or more other peptides. Other linkage sequencesincluded are leucine enkephalin derivatives such as Tyr-Gly-Gly-Phe-Leu,and the like.

[0046] Another preferred type of biocleavable linkage is any “hindered”or “protected” disulfide bond that sterically inhibits attack fromthiolate ions. Examples of such protected disulfide bonds are found inthe coupling agents: S-4-succinnimdyl-oxycarbonyl-ø-methyl benzylthiosulfate (SMBT) and4succninidyloxycarbonyl-ø-methyl-ø-(2-pyridyldithio) toluene (SMPT).Another useful coupling agent resistant to reduction is SPDB disclosedby Worrell, et al., Anticancer Drug Design 1:179-188 (1986). Alsoincluded are certain aryldithio thioimidates, substituted with a methylor phenyl group adjacent to the disulfide, which include ethyl S-acetyl3-mercaptobutyrothioimidate (M-AMPT)and 3-(4-carboxyamidophenyldithio)proprionthioimidate (CDPT), disclosed by S. Arpicco, et al., Bioconj.Chem. 8 (3):327-337 (1997). Another preferred category is certainlinkages or bonds subject to hydrolysis that include various aldehydebonds with amino or sulfhydryl groups. Also included are amide bondssuch as when N-hydroxysuccinimide ester (NHS ester) reacts with amines.

[0047] Biorecognition Molecules.

[0048] For the purposes of this invention, biorecognition molecules arethose that bind to a specific biological substance or site. Thebiological substance or site is considered the “target” of thebiorecognition molecule that binds to it. In the prior art, many drugsare “targeted” by coupling them to a biorecognition molecule that has aspecific binding affinity for the cells, tissue or organism that thedrug is intended for. For targeting a drug or other active agent in thisinvention, a biorecognition molecule is coupled to a cyclodextrinpolymer carrier that is used to entrap the drug. Examples ofbiorecognition molecules are described below.

[0049] Ligand.

[0050] A ligand functions as a type of biorecognition molecule definedas a selectively bindable material that has a selective (or specific),affinity for another substance. The ligand is recognized and bound by ausually, but not necessarily, larger specific binding body or “bindingpartner”, or “receptor”. Examples of ligands suitable for targeting areantigens, haptens, biotin, biotin derivatives, lectins, galactosamineand fucosylamine moieties, receptors, substrates, coenzymes andcofactors among others.

[0051] When applied to the cyclodextrin polymers of this invention, aligand includes an antigen or hapten that is capable of being bound by,or to, its corresponding antibody or fraction thereof. Also included areviral antigens or hemagglutinins and neuraminidases and nucleocapsidsincluding those from any DNA and RNA viruses, AIDS, HIV and hepatitisviruses, adenoviruses, alphaviruses, arenaviruses, coronaviruses,flaviviruses, herpesviruses, myxoviruses, oncornaviruses, papovaviruses,paramyxoviruses, parvoviruses, picornaviruses, poxviruses, reoviruses,rhabdoviruses, rhinoviruses, togaviruses and viroids; any bacterialantigens including those of gram-negative and gram-positive bacteria,acinetobacter, achromobacter, bacteroides, clostridium, chlamydia,enterobacteria, haemophilus, lactobacillus, neisseria, staphyloccus, andstreptoccocus; any fungal antigens including those of aspergillus,candida, coccidiodes, mycoses, phycomycetes, and yeasts; any mycoplasmaantigens; any rickettsial antigens; any protozoan antigens; any parasiteantigens; any human antigens including those of blood cells, virusinfected cells, genetic markers, heart diseases, oncoproteins, plasmaproteins, complement factors, rheumatoid factors. Included are cancerand tumor antigens such as alpha-fetoproteins, prostate specific antigen(PSA) and CEA, cancer markers and oncoproteins, among others.

[0052] Other substances that can function as ligands for biorecognitionare certain vitamins (i.e. folic acid, B₁₂), steroids, prostaglandins,carbohydrates, lipids, antibiotics, drugs, digoxins, pesticides,narcotics, neuro-transmitters, and substances used or modified such thatthey function as ligands. Most preferred are certain proteins or proteinfragments (i.e. hormones, toxins), and synthetic or natural polypeptideswith cell affinity. Ligands also include various substances withselective affinity for ligators that are produced through recombinantDNA, genetic and molecular engineering. Except when stated otherwise,ligands of the instant invention also include the ligands as defined byK. E. Rubenstein, et al, U.S. Pat. No. 3,817,837 (1974).

[0053] Ligator.

[0054] A ligator functions as a type of biorecognition molecule definedfor this invention as a specific binding body or “partner” or“receptor”, that is usually, but not necessarily, larger than the ligandit can bind to. For the purposes of this invention, it is a specificsubstance or material or chemical or “reactant” that is capable ofselective affinity binding with a specific ligand. A ligator can be aprotein such as an antibody, a nonprotein binding body or a “specificreactor.”

[0055] When applied to this invention, a ligator includes an antibody,which is defined to include all classes of antibodies, monoclonalantibodies, chimeric antibodies, Fab fractions, fragments andderivatives thereof. Under certain conditions, the instant invention isalso applicable to using other substances as ligators. For instance,other ligators suitable for targeting include naturally occurringreceptors, any hemagglutinins and cell membrane and nuclear derivativesthat bind specifically to hormones, vitamins, drugs, antibiotics, cancermarkers, genetic markers, viruses, and histocompatibility markers.Another group of ligators includes any RNA and DNA binding substancessuch as polyethylenimine (PEI) and polypeptides or proteins such ashistones and protamines.

[0056] Other ligators also include enzymes, especially cell surfaceenzymes such as neuraminidases, plasma proteins, avidins, streptavidins,chalones, cavitands, thyroglobulin, intrinsic factor, globulins,chelators, surfactants, organometallic substances, staphylococcalprotein A, protein G, ribosomes, bacteriophages, cytochromes, lectins,certain resins, and organic polymers.

[0057] Preferred biorecognition molecules also include varioussubstances such as any proteins, protein fragments or polypeptides withaffinity for the surface of any cells, tissues or microorganisms thatare produced through recombinant DNA, genetic and molecular engineering.For instance, any suitable membrane transfer proteins such as TAT, fromHIV virus.

[0058] Nucleic Acid Biorecognition Molecules.

[0059] For the purposes of this invention, certain nucleic acids canfunction as biorecognition molecules. A nucleic acid biorecognitionmolecule is defined as any nucleic acid sequence from any source that iscoupled to the cyclodextrin polymer carrier for targeting a specifictype of microbe, cell or tissue.

[0060] Preferred nucleic acid biorecognition molecules are sequencesthat “recognize” or hybridize with a disease-specific nucleic acidsequence (i.e. mRNA or DNA) found within a target cell, as described byZ. Ma, et al., PNAS 97, 11159-11163 (2000). A CD carrier containing asuitable active agent or, a CD catalytic agent of this invention, wouldbe coupled to a suitable nucleic acid that recognized a disease-specificsequence in a cell.

[0061] Nucleic acid biorecognition molecules include all types of RNA,all types of DNA, and oligonucleotides including probes and primers usedin the polymerase chain reaction (PCR), or DNA sequencing. Also includedare synthetic nucleic acid polymers and peptide nucleic acids (PNA)disclosed by Egholm, et al, Nature 365:566-568(1993) and referencestherein, including PNA clamps (Nucleic Acids Res. 23:217(1995)). Alsoincluded are DNA and/or RNA fragments, and derivatives from any tissue,cells, nuclei, chromosomes, cytoplasm, mitochondria, ribosomes, andother cellular sources.

[0062] Cyclodextrin.

[0063] A cyclodextrin (CD), is an oligosaccharide composed of glucosemonomers coupled together to form a conical hollow molecule with ahydrophobic interior or cavity. The cyclodextrins of the instantinvention can be any suitable cyclodextrin, including alpha-, beta-, andgamma-cyclodextrins, and their combinations, analogs, isomers, andderivatives. They function as components in the synthesis of thecyclodextrin polymer carriers of the instant invention.

[0064] In describing this invention, references to a cyclodextrin“complex”, means a noncovalent inclusion complex. An inclusion complexis defined herein as a cyclodextrin functioning as a “host” molecule,combined with one or more “guest” molecules that are contained or bound,wholly or partially, within the hydrophobic cavity of the cyclodextrinor its derivative.

[0065] Most preferred are cyclodextrin dimers, trimers and polymerscontaining cyclodextrin derivatives such as carboxymethyl CD, glucosylCD, maltosyl CD, hydroxypropyl cyclodextrins (HPCD), 2-hydroxypropylcyclodextrins, 2,3-dihydroxypropyl cyclodextrins (DHPCD),sulfobutylether cyclodextrins (SBECD), ethylated and methylatedcyclodextrins.

[0066] Also preferred are oxidized cyclodextrins that provide aldehydesand any oxidized forms of any cyclodextrn polymers or derivatives thatprovide aldehydes. Some examples of suitable derivatives are disclosedby Pitha, J., et al, J. Pharm. Sci. 75, 165-167 (1986) and Pitha, J., etal, Int. J. Pharmaceut. 29, 73-82 (1986).

[0067] Also preferred are any amphiphilic CD derivatives such as thosedisclosed by Y. Kawabata, et al., Chem. Lett. p1933 (1986), K Chmurski,et al., Langmuir 12, 4046 (1996), P. Zhang, et al., Tetr. Lett. 32,No.24, 2769 (1991), P. Zhang, et al., J. Phys. Org. Chem. 5, 518 (1992),M. Tanaka, et al., Chem. Lett. p1307 (1987), S. Taneva, et aL, Langmuir5, 111 (1989), M. Weisser, et al., J. Phys. Chem. 100, 17893 (1996), L.A. Godinez, et al., Langmuir 14, 137 (1998) and D. Duchene,“International Pharmaceut. Applic. of Cyclodextrins Conference”,Lawrence, Kans., USA, June 1997.

[0068] Also included are altered forms, such as crown ether-likecompounds prepared by Kandra, L., et al, J. Inclus. Phenom. 2, 869-875(1984), and higher homologues of cyclodextrins, such as those preparedby PulLey, et al, Biochem. Biophys. Res. Comm. 5, 11 (1961). Some usefulreviews on cyclodextrins are: Atwood J. E. D., et al, Eds., “InclusionCompounds”, vols. 2 & 3, Academic Press, NY (1984); Bender, M. L., etal, “Cyclodextnin Chemistry”, Springer-Verlag, Berlin, (1978) andSzejtli, J., “Cyclodextrins and Their Inclusion Complexes”, AkademiaiKiado, Budapest, Hungary (1982). These references, including referencescontained therein, are applicable to the synthesis of the preparationsand components of the instant invention and are hereby incorporatedherein by reference.

[0069] Cyclodextrin Dimers, Trimers and Polymers.

[0070] For this invention, individual cyclodextrin (CD-monomer)derivatives function as the primary building structures, or components,or units used to synthesize the water-soluble (or colloidal)cyclodextrin polymer carriers. Also, certain preferred CD dimers, andtrimers of this invention are not used as building units for polymersand can function as drug carriers or excipients without additionalcrosslinking.

[0071] A cyclodextrin dimer is defined as two cyclodextrin moleculescovalently coupled or cross-linked together to enable cooperativecomplexing with a guest molecule. Examples of some CD dimers that can bederivatized and used in the drug carriers of this invention, aredescribed by; Breslow, R., et al, Amer. Chem. Soc. 111, 8296-8297(1989); Breslow, R., et a, Amer. Chem. Soc. 105, 1390 (1983) and Fujita,K., et al, J. Chem. Soc., Chem. Commun., 1277 (1984).

[0072] A cyclodextrin trimer is defined as three cyclodextrin moleculescovalently coupled or cross-linked together to enable cooperativecomplexing with a guest molecule. A cyclodextrin polymer is defined as aunit of more than three cyclodextrin molecules covalently coupled orcross-linked together to enable cooperative complexing with severalguest molecules.

[0073] A CD-block is defined as a CD dimer, trimer or polymer that isused as the primary component, or unit (i.e. building block) foradditional crosslinking with other CD dimers, trimers or polymers tosynthesize a CD polymer carrier. Generally this involves at least twocrosslinking steps, where first the CD-blocks are prepared bycrosslinking CD monomers and derivatized or activated for subsequentcoupling. Then the CD-blocks are crosslinked in a second reaction toentrap the active agent in the final CD carrier composition. An exampleof this method is given below.

[0074] For this invention, preferred cyclodextrin dimer, trimer andpolymer units or blocks are synthesized by covalently coupling throughchemical groups such as through coupling agents generally not to exceed50 angstroms in spacer length. The synthesis of preferred cyclodextrindimer, trimer and polymer units or CD blocks does not include the use ofproteins or other “intermediate coupling substances” (defined below),which can be incorporated during final synthesis of the cyclodextrinpolymer carrier. Cooperative complexing means that in situations wherethe guest molecule is large enough, the member cyclodextrins of the CDdimer, trimer or polymer can each noncovalently complex with differentparts of the same guest molecule, or with smaller guests, alternatelycomplex with the same guest.

[0075] The prior art has disclosed dimers and polymers comprised ofcyclodextrins of the same size. An improved cyclodextrin dimer, trimeror polymer comprises combinations of different sized cyclodextrins tosynthesize these units. These combinations may more effectively complexwith guest molecules that have heterogeneous complexing sites.Combinations for this invention can include the covalent coupling of analpha CD with a beta CD, an alpha CD with a gamma CD, a beta CD with agamma CD and polymers with various ratios of alpha, beta and gammacyclodextrins.

[0076] Cyclodextrin Polymer Carrier.

[0077] A water-soluble (or colloidal) cyclodextrin polymer carrier is anew composition provided by the instant invention. It is defined hereinas a polymer of cross-linked cyclodextrin derivatives that has thedistinguishing property of having incorporated a drug or other activeagent as a “captured guest”. The “capture” of the guest stabilizes thecarrier complex and overcomes the problem in the prior art of the CDhost and guest molecules separating by diffusion. Generally, the agenthas also formed a noncovalent “inclusion complex”, or “inclusioncompound” with the cyclodextrins of the polymer.

[0078] Self-Assembled Cyclodextrin Polymer Carrier.

[0079] A self-assembled, or self-coupled or auto-assembled cyclodextrinpolymer carrier is a new composition provided by the instant invention.It is defined herein as a water-soluble (or colloidal) polymer ofcross-linked cyclodextrin derivatives that has the distinguishingproperty of having incorporated a drug or other active agent as a“captured guest”. The “capture” of the guest also includes stabilizingthe carrier complex and overcomes the problem in the prior art of the CDhost and guest molecules separating by diffusion.

[0080] Captured Guest Cyclodextrin Polymer Carrier.

[0081] In a preferred embodiment the capturing is accomplished throughcomplete physical entrapment by the water-soluble (or colloidal) CDpolymer carrier. In this embodiment, “completely entrapped” means that acaptured guest is not covalently coupled to the polymer but isphysically entrapped by the covalently cross-linked polymer ofcyclodextrin molecules so that no significant amount of active agent canleave the polymer by diffusion or extraction. Completely entrappedsmaller guest molecules such as drugs and ligands are suitably“non-diffusable”, by being entrapped wholly within the polymer.

[0082] Completely entrapped larger guests such as proteins,polypeptides, and nucleic acids (DNA, RNA, oligonucleotides) aresuitably non-diffusable by being entrapped wholly or partially so thatthe guest and polymer still cannot separate by diffusion. Completelyentrapped guests cannot escape until the polymer itself has beendegraded or the covalent cross-link bonds are cleaved. In thisembodiment, essentially all possible exit routes for the guest to leavethe polymer have been closed by cross-linking. Therefore, additionalguest molecules (of that size or larger) cannot enter the dosed polymerto be added to the cyclodextrin polymer carrier.

[0083] This is made possible through the unique method for synthesizingthe cyclodextrin polymer carriers of the instant invention. Thedistinguishing principal of the method is that the guest molecules arecompletely entrapped during polymerization or during the finalcross-linking step of making the polymer carrier. Initially, guestmolecules are mixed with the “open” components of the cyclodextrinpolymer, which may comprise individual cyclodextrins (or derivatives),cyclodextrin dimers, trimers or an open cyclodextrin polymer. An opencyclodextrin polymer means that the polymer is only partiallycross-linked so that guest molecules can enter or associate with thepolymer by diffusion and form complexes with member cyclodextrins. Inthe final synthesis step of the polymer carrier, the polymer is closedby additional covalent cross-linking which completely entraps the guestsas defined previously.

[0084] Controlled Release.

[0085] For this invention, controlled release is defined as the releaseof a captured guest from the CD polymer carrier only by cleavage ofcertain covalent linkages that were used to synthesize the carrier. Thisdefinition specifically excludes release by diffusion until saidlinkages are cleaved.

[0086] Targeted Cyclodextrin Polymer Carriers.

[0087] A targeted cyclodextrin (CD) polymer carrier is an embodiment ofthis invention composed of a water-soluble (or colloidal) cyclodextrinpolymer carrier, or derivative described herein, that has abiorecognition molecule covalently coupled to its surface. However, thebiorecognition molecule is not an inclusion complex within thecyclodextrin carrier. The carrier is thereby targeted through thespecific binding properties of the biorecognition molecule coupled tothe surface.

[0088] During the coupling, the functions of the biorecognition moleculeand the targeted CD polymer carrier are not irreversibly or adverselyinhibited. Preferably, the biorecognition molecule maintains specificbinding properties that are functionally identical or homologous tothose it had before coupling. Preferably, the biorecognition molecule iscoupled through a suitable spacer to avoid steric hindrance.

[0089] Targeted cyclodextrin polymer carriers coupled to avidin andstreptavidin are useful for subsequent noncovalent coupling to anysuitable biotinylated substance. Similarly, cyclodextrin polymercarriers coupled to antibody can be noncovalently coupled to anotherantibody, or to a nucleic acid or other suitable substance that has theappropriate biorecognition properties. Another useful cyclodextrincarrier comprises protein A, protein G, or any suitable lectin orpolypeptide that has been covalently coupled to a cyclodextrin polymercarrier.

[0090] Biocleavable Cross-Linking Agent.

[0091] A biocleavable cross-linking agent comprises a new compositionfor facilitating the synthesis of drug carriers with controlled release.In one embodiment it is comprised of a biocleavable sequence of aminoacids between suitable compounds that comprise or contain amino-reactiveor thiol-reactive coupling groups at each end for direct coupling toamino or sulfhydryl groups on an active agent or polymer. Using theconvention of starting with the amino or “N” terminus on the left andthe carboxyl or “C” terminus on the right, some examples are: anypolypeptide compounds that contain the sequence Phe-Leu, Leu-Phe orPhe-Phe, such as Gly-Phe-Leu, Gly-Leu-Phe, Gly-Phe-Leu-Gly,Gly-Phe-Leu-Phe-Gly and Gly-Phe-Phe-Gly, Gly-Phe-Leu-Gly-Lys,Lys-Gly-Phe-Leu-Gly-Lys and others that have either of the Gly residuessubstituted for one or more other peptides. Also included are leucineenkephalin derivatives such as Tyr-Gly-Gly-Phe-Leu. A preferredembodiment comprises a polypeptide with a biocleavable sequence asdescribed, and also includes terminal compounds with coupling groupssuch as N-succinimidyl N-maleimidyl, iodoacetal, bromoacetal oxirane orimidoester coupling groups on each end.

[0092] For acid-labile biocleavable cross-linking agents, one embodimentcomprises a bifunctional coupling agent with a hydrazone linkageincorporated into it. For instance, it would comprise a hydrazonelinkage between suitable compounds comprising aliphatic chains oraromatic groups that have terminal N-succinimidyl, N-maleimidyl,p-nitrophenyl ester (ONp), iodoacetal, bromoacetal oxirane or imidoestercoupling groups on each end. One example for synthesizing an acid-labilebiocleavable coupling agent is to first react an excess ofhydrazinobenzoic acid with glutaraldehyde to couple one hydrazinobenzoicacid at each end of the dialdehyde. This produces hydrazone linkageswith terminal carboxyl groups at each end. The terminal carboxyl groupsare then converted to N-succinimidyl ester groups.

[0093] Coupling.

[0094] For the instant invention, two distinct types of coupling aredefined. One type of coupling can be through noncovalent, “attractive”binding as with a guest molecule and cyclodextrin, antigen and antibodyor biotin and avidin. Noncovalent coupling is binding between substancesthrough ionic or hydrogen bonding or van der waals forces, and/or theirhydrophobic or hydrophilic properties.

[0095] Unless stated otherwise, the preferred coupling used in theinstant invention is through covalent, electron-pair bonds or linkages.Many methods and agents for covalently coupling (or crosslinking)cyclodextrins and cyclodextrin derivatives are known and, withappropriate modification, can be used to couple the desired substancesthrough their “functional groups” for use in this invention.

[0096] Where stability is desired, the preferred linkages are amidebonds, peptide bonds, ether bonds, and thio ether bonds, among others.

[0097] Functional Group.

[0098] A functional group is defined here as a potentially reactive siteon a substance where one or more atoms are available for covalentcoupling to some other substance. When needed, functional groups can beadded to various substances through derivatization or substitutionreactions.

[0099] Examples of functional groups are aldehydes, allyls, amines,amides, azides, carboxyls, carbonyls, epoxys (oxiranes), ethynyls,hydroxyls, ketones, certain metals, nitrenes, phosphates, propargyls,sulfhydryls, sulfonyls, phenolic hydroxyls, indoles, bromines,chlorines, iodines, and others. The prior art has shown that most, ifnot all of these functional groups can be incorporated into or added tocyclodextrins, biorecognition molecules, drugs, nucleic acids andsupport materials.

[0100] Cross-Linking or Coupling Agent.

[0101] A coupling agent (or cross-linking agent), is defined as achemical substance that produces and/or reacts with functional groups ona substance to produce covalent coupling, cross-linking, or conjugationwith that substance. Because of the stability of covalent coupling, thisis the preferred method. Depending on the chemical makeup or functionalgroup on the cyclodextrin, nucleic acid, or biorecognition molecule, theappropriate coupling agent is used to provide the necessary activefunctional group or to react with the functional group. In certainpreparations of the instant invention, coupling agents are needed thatprovide a spacer between cross-linked cyclodextrins or betweencyclodextrin and a biorecognition molecule to overcome steric hindrance.Preferably, the spacer is a substance of 4 or more carbon atoms inlength and can include aliphatic, aromatic and heterocyclic structures.

[0102] With appropriate modifications by one skilled in the art, thecoupling methods referenced in U.S. Pat. No. 6,048,736 andPCr/US99/30820, including references contained therein, are applicableto the synthesis of the preparations and components of the instantinvention and are hereby incorporated by reference, herein:

[0103] Examples of energy activated coupling or cross-linking agents areultraviolet (UV), visible and radioactive radiation that can promotecoupling or crosslinking of suitably derivatized cyclodextrins. Examplesare photochemical coupling agents disclosed in U.S. Pat. No. 4,737,454,among others. Also useful in synthesizing components of the instantinvention are enzymes that produce covalent coupling such as nucleicacid polymerases and ligases, among others.

[0104] In some preferred aspects of this invention, cyclodextrin dimers,trimers and polymers are first prepared for use as the primarycomponents, or CD-blocks to synthesize the cyclodextrin polymercarriers. Useful derivatizing and/or coupling agents for preparingCD-blocks are bifunctional, trifunctional or polyfunctional crosslinkingagents that will covalently couple to the hydroxyl groups ofcyclodextrin. Some preferred examples are oxiranes such asepichlorohydrin, epoxides such as 1,4 butanediol diglycidyl ether (BDE),glycerol diglycidyl ether (GDE), trimethylolpropane triglycidyl ether(TMTE), glycerol propoxylate triglycidyl ether (GPNI), 1,3-butadienediepoxide, triphenylolmethane triglycidyl ether, 4,4′-methylenebis(N,N-diglycidylaniline), tetraphenylolethane glycidyl ether, bisphenol Adiglycidyl ether, bisphenol A propoxylate diglycidyl ether, bisphenol Fdiglycidyl ether, cyclohexanedimethanol diglycidyl ether, 2,2′-oxybis(6-oxabicyclo[3.1.0] hexane), polyoxyethylene bis(glycidyl ether),resorcinol diglycidyl ether, ethylene glycol diglycidyl ether (EGDE) andlow molecular weight forms of poly(ethylene glycol) diglycidyl ethers orpolypropylene glycol) diglycidyl ethers, among others.

[0105] Other preferred derivatizing and/or coupling agents for hydroxylgroups are various disulfonyl compounds such as benzene-1,3-disulfonylchloride and 4,4′-biphenyl disulfonyl chloride and also divinyl sulfone,among others.

[0106] Most preferred coupling agents are also chemical substances thatcan provide the bio-compatible linkages for synthesizing thecyclodextrin polymer carriers of the instant invention. Covalentcoupling or conjugation can be done through functional groups usingcoupling agents such as glutaraldehyde, formaldehyde, cyanogen bromide,azides, p-benzoquinone, maleic or succinic anhydrides, carbodiimides,epichlorohydrin, ethyl chloroformate, dipyridyl disulfide andpolyaldehydes.

[0107] Also most preferred are derivatizing and/or coupling agents thatcouple to thiol groups (“thiol-reactive”) such as agents with anymaleimide, vinylsulfonyl, bromoacetal or iodoacetal groups, includingany bifunctional or polyfunctional forms. Examples arem-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),succinimidyl-4-(N-maleimidomethyl)cydohexane-1-carboxylate (SMCC),succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB),dithiobis-N-ethylmaleimide (DTEM), 1,1′-(methylenedi-4,1-phenylene)bismaleimide (MPBM), o-phenylenebismaleimide, N-succinimidyl iodoacetate(SIA), N-succinimidyl-(4-vinylsulfonyl) benzoate (SVSB), andTris-(2-maleimidoethyl) amine (TMEA) among others.

[0108] Other coupling groups or agents useful in the instant inventionare: p-nitrophenyl ester (ONp), bifunctional imidoesters such asdimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethylsuberimidate (DMS), methyl 4-mercaptobutyrimidate, dimethyl3,3′-dithiobis-propionimidate (DTBP), and 2-iminothiolane (Traut'sreagent);

[0109] bifunctional NHS esters such as disuccinimidyl suberate (DSS),bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), disuccinimidyl(N,N′-diacetylhomocystein) (DSAH), disuccinimidyl tartrate (DST),dithiobis(succinimidyl propionate) (DSP), and ethylene glycolbis(succinimidyl succinate) (EGS), including various derivatives such astheir sulfo-forms;

[0110] heterobifunctional reagents such asN-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), p-azidophenacylbromide, p-azidophenylglyoxal, 4-fluoro-3-nitrophenyl azide (FNPA),N-hydroxysuccinimidyl-4azidobenzoate (HSAB), methyl-4-azidobenzoimidate(MABI), p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate,N-succinimidyl-6(4′-azido-2′-nitrophenylamino) hexanoate (Lomant'sreagent II), N-succinimidyl (4-azidophenyldithio)propionate (SADP),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andN-(4-azidophenylthio)phthalimide (APTP), including various derivativessuch as their sulfo- forms;

[0111] homobifunctional reagents such as1,5-difluoro-2,4-dinitrobenzene,4,4′-difluoro-3,3′-dinitrophenylsulfone,4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS),p-phenylenediisothiocyanate (DITC), carbonylbis(L-methioninep-nitrophenyl ester), 4,4′-dithiobisphenylazide anderythritolbiscarbonate, including various derivatives such as theirsulfo- forms;

[0112] photoactive coupling agents such asN-5-azido-2-nitrobenzoylsuccinimide (ANB-NOS), p-azidophenacyl bromide(APB), p-azidophenyl glyoxal (APG), N-(4-azidophenylthio)phthalimide(APTP), 4,4′-dithio-bis-phenylazide (DTBPA), ethyl4-azidophenyl-1,4-dithiobutyrimidate (EADB), 4-fluoro-3-nitrophenylazide (FNPA), N-hydroxysuccinimidyl-4-azidobenzoate (HSAB),N-hydroxysuccinimidyl-4-azidosalicylic add (NHS-ASA),methyl-4-azidobenzoimidate (MABI),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),2-diazo-3,3,3-trifluoropropionyl chloride,N-succinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate (SANPAH)N-succinimidyl(4azidophenyl)1,3′-dithiopropionate (SADP),sulfosuccinimidyl-2-(m-azido-o-nitobenzamido)-ethyl-1,3′-dithiopropionate(SAND), sulfosuccinimidyl(4-azidophenyldithio)propionate (Sulfo-SADP),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(Sulfo-SANPAH),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(SASD), and derivatives and analogs of these reagents, among others. Thestructures and references for use are given for many of these reagentsin, “Pierce Handbook and General Catalog”, Pierce Chemical Co.,Rockford, Ill., 61105.

[0113] Intermediate Coupling Substance.

[0114] In addition to covalently coupling directly through functionalgroups of cyclodextrin derivatives to synthesize water-soluble (orcolloidal) polymers, it is also useful to include an intermediatesubstance or “intermediate”. By definition, intermediate substancesfunction as bio-compatible intermediates in being suitablynonimmunogenic and nonallergenic. Although intermediate substances maybe degraded biologically, they are “biologically neutral” in that theyessentially lack specific binding properties or biorecognitionproperties in their application.

[0115] The intermediate can function as a “spacer” (e.g. “spacer arm” ofO'Carra, P., et al, FEBS Lett. 43, 169 (1974)), between the cyclodextrinderivatives being covalently coupled to overcome steric hindrance ofsubsequent binding reactions. The intermediate can function as a polymer“backbone” to which many cyclodextrin dimers, trimers or polymers arecovalently coupled to form a larger polymer. The intermediate can beincluded with cyclodextrin derivatives as another monomer to becopolymerized with the cyclodextrin derivatives (i.e. heteropolymer), toprovide improved structural properties, increase solubility or lowertoxicity.

[0116] The intermediate substance may also provide the advantage ofadditional coupling sites and thereby increase the number of covalentlycoupled cyclodextrin derivatives within a polymer carrier. Theintermediate can also introduce certain other desirable properties, suchas a positive or negative net charge, more efficient light energytransfer for photodynamic therapy. The desired biorecognition moleculeor other substance can be coupled to the available sites on theintermediate substance and is thereby coupled indirectly to thewater-soluble cyclodextrin polymer carrier of the instant invention.

[0117] Examples of such biologically neutral intermediate couplingsubstances are certain proteins, polypeptides, polyamino acids, serumalbumins, glycoproteins, lipoproteins, nucleic acid polymers, DNA, RNA,amino sugars, glucosamines, polysaccharides, lipopolysaccharides, aminopolysaccharides, polyglutamic acids, polylysines, polyacrylamides,nylons, poly(allylamines), lipids, glycolipids and suitable syntheticpolymers, especially biopolymers, resins and surfactants, as well assuitable derivatives of these substances. Also included as suitableintermediate coupling substances are the polymers disclosed in U.S. Pat.No. 4,645,646. Also preferred as intermediates areN-(2-hydroxypropyl)methacrylamide (HPMA), HPMA derivatives, polycyanoacrylates such as poly(butyl cyanoacrylate), poly(isobutyl orisohexyl cyanoacrylate), polyethylene glycol (PEG), any PEG derivatives,poly (D,L-lactic-coglycolic acid) (PLGA), PLGA derivatives, dendrimersand poly (D,L-lactide)-block-methoxy-polyethylene glycol (Diblock).

[0118] Various materials may be incorporated into the components of theinstant invention to produce new inventions with unexpected propertiesfor use in certain applications. For instance, the addition of ferrousor magnetic particles may be used to give cyclodextrin polymer carriersand other types of polymers (i.e. HPMA, PEG), magnetic properties(Ithakissios, D. S., Clin. Chim. Acta 84(1-2), 69-84, 1978). This wouldbe useful for various in vivo manipulations such as using magneticfields to localize or concentrate a magnetic polymer drug carrier in aspecific part of the body. Also, the magnetic particles may be used totrigger a cytotoxic effect on cancer cells such as by vibrating themwith alternating magnetic fields.

[0119] CD Guest-Linked Agent.

[0120] A cyclodextrin guest-linked agent or “CD guest-linked agent”comprises a new invention for facilitating the noncovalent coupling ofCD polymers with drugs, proteins, DNA, ODNs and other active agents. Inone embodiment a CD guest-linked agent or simply “guest-linked agent”(GLA), is comprised of an active agent covalently coupled to one or moremolecules capable of forming an inclusion complex (“complex”) with aspecific CD or CD polymer. The inclusion complex between the CD and theGLA is preferably of higher affinity than between the CD and the activeagent alone. The major advantage is that an active agent that is noteasily complexed with a cyclodextrin or CD polymer is thereby moreeasily complexed or linked when it is a GLA. This then provides a uniquemethod for entrapping an active agent as a guest-linked agent in a CDpolymer.

[0121] Preferably the GLA includes guest molecules (the CD linker orcoupler), that have high affinity for a specific cyclodextrin.Generally, these high affinity guest molecules ate those that fit moresnugly or closely within the hydrophobic cavity of either alpha, beta orgamma cyclodextrin. The most preferred guest molecules have associationconstants of 1×10³ M⁻¹ or more. For example, preferred guest moleculesfor beta cyclodextrin include any suitable adamantane analogs orderivatives such as adamantane acetate (AAC), adamantane carboxylate(AC), 1-homoadamantanes (1-HAC), 3-homoadamantanes (3-HAC),3-noradamantane carboxylate (NAC), norbornane acetate (NBA),1-bicyclo[2.2.1]octanecarboxylate, 1-bicyclo-[2.2.1]heptane carboxylate,1-bicyclo[2.2.1]heptene carboxylate; any suitable analogs or derivativesof cyclohexane such as cyclohexanecarboxylate, cyclohexane acetic acid;any suitable analogs or derivatives of cyclopentane such ascyclopentanecarboxylate; any suitable analogs or derivatives of benzene;any suitable analogs or derivatives of camphor; among others.

[0122] For example, the GLA invention can be used to complex a drug suchas doxorubicin (DOX) with a CD polymer. The drug is first derivatized toproduce an active form with an amino-coupling group such as NHS ester orp-nitrophenyl ester (ONp) as described by P. Rejmanova, et al.,Makromol. Chem. 178, 2159 (1977). DOX is suitably derivatized to providean ONOp group as described by V. Omelyanenko, et al., J. Controlled Rel.53, 25-37 (1998). A GLA is then synthesized by covalently coupling theONp-DOX derivative to a suitable amino-derivatized adamantane such as1-aminoadamantane, 1-adamantane methyamine, or 1-adamantane carboxamide,among others.

[0123] Conversely, any suitable active agent can be derivatized toproduce an active form that provides an amino functional group forcoupling. A GLA is then synthesized by covalently coupling the activeagent through the amino group to an adamantane derivative with anamino-coupling group on it. Such adamantane derivatives include1-adamantane carbonyl chloride, 1-adamantane isothiocyanate, oradamantane derivatized to provide an NHS ester or ONp, among others.

[0124] Also, any suitable active agent can be derivatized to produce anactive form that provides a sulfhydryl functional group for coupling. AGLA is then synthesized by covalently coupling the active agent throughthe sulfhydryl group to an adamantane derivative with a thiol- orsulfhydryl-coupling group on it. Such adamantane derivatives include1-adamantane carbonyl chloride, iodo adamantane, or adamantanederivatized to provide a maleimidyl or bromoacetyl group. Also, suitablesulfhydryl-derivatized adamantane can be used to couple to an activeagent with available sulfhydryl groups through disulfide coupling.

[0125] Also, when any suitable active agent is derivatized to produce anactive form that provides an amino, or sulfhydryl functional group forcoupling. A GLA is then synthesized by covalently coupling the activeagent to an adamantane derivative with an amino, or thiol, or hydroxylfunctional group available. Such coupling is done through any suitablecrosslinking agent reactive with the functional groups available on theactive agent and the guest (“CD linker”). In any case, the newlysynthesized CD guest-linked agent can then be entrapped by mixing withthe CD dimers, CD trimers or CD polymer carriers described herein.

[0126] A new, preferred guest-linked agent has dimer, trimer or smallpolymer adamantanes (or other suitable guests) coupled at single siteson the drug, nucleic acid or other active agent. This provides a newclass of CD linkers that provides a higher number of complexing gueststhan is possible to couple to smaller active agents such as drugs andODNs with fewer coupling sites. This new CD linker is also more easilycomplexed with dimers, or trimers or polymers of beta cyclodextrin,making the active agent more easily solubilized and the inclusioncomplexes more stable than with individual adamantanes coupled to theactive agent.

[0127] Preferably, any of the coupling systems used to synthesize a GLAalso include a biocleavable linkage, described herein, to provide forcontrolled release of the active agent if desired.

EXAMPLES OF THE BEST MODES FOR CARRYING OUT THE INVENTION

[0128] In the examples to follow, percentages are by weight unlessindicated otherwise. During the synthesis of the compositions of theinstant invention, it will be understood by those skilled in the art oforganic synthesis, that there are certain limitations and conditions asto what compositions will comprise a suitable carrier and may thereforebe prepared mutatis mutandis. It will also be understood in the art ofcyclodextrins that there are limitations as to which drugs and otheragents can be used to form inclusion complexes with certaincyclodextrins.

[0129] Specifically, it is known that smaller, alpha cyclodextrins arepreferably used to complex with the smaller drugs or active agents.Whereas larger cyclodextrins are less limited, except that a “close fit”is generally preferred for stronger complexing affinity.

[0130] The terms “suitable” and “appropriate” refer to synthesis methodsknown to those skilled in the art that are needed to perform thedescribed reaction or procedure. In the references to follow, themethods are hereby incorporated herein by reference. For example,organic synthesis reactions, including cited references therein, thatcan be useful in the instant invention are described in “The MerckIndex”, 9, pages ONR-1 to ONR-98, Merck & Co., Rahway, N.J. (1976), andsuitable protective methods are described by T. W. Greene, “ProtectiveGroups in Organic Synthesis”, Wiley-Interscience, NY (1981), amongothers. For synthesis of nucleic acid probes, sequencing andhybridization methods, see “Molecular Cloning”, 2nd edition, T.Maniatis, et al, Eds., Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.(1989).

[0131] All reagents and substances listed, unless noted otherwise, arecommercially available from Aldrich Chemical Co., WI 53233; SigmaChemical Co., Mo. 63178; Pierce Chemical Co., IL. 61105; Eastman KodakCo., Rochester, N.Y.; Pharmatec Inc., Alachua Fla. 32615; and ResearchOrganics, Cleveland, Ohio. Or, the substances are available or can besynthesized through referenced methods, including “The Merck Index”, 9,Merck & Co., Rahway, N.J. (1976).

[0132] Additional references cited in U.S. Pat. No. 6,048,736 andPCT/US99/30820, are hereby incorporated herein by reference.

Cyclodextrin Polymer Carriers

[0133] The purpose is to provide a water-soluble (or colloidal)cyclodextrin polymer carrier that has an active agent completelyentrapped. For synthesis, the general approach is; (1) to produce ormodify or protect, as needed, one or more functional or coupling groupson the cyclodextrin components, consisting of cyclodextrins, or opendimers, trimers or polymers; (2) combine under appropriate conditions, aminimum of 2 of the cyclodextrin components with a drug or active agentto produce a noncovalent inclusion complex and (3) if needed, usingvarious coupling methods cross-link the cyclodextrin components toproduce a polymer that completely entraps the drug within thecyclodextrin polymer. In certain applications, the complex between CDcomponents and active agent produced in step 2 will be suitable forcarrying the active agent.

[0134] Also, as described below, the cyclodextrin polymer carrier may besuitably derivatized to include other useful substances and/or chemicalgroups (e.g. biorecognition molecules, antenna, and catalyticsubstances), to perform a particular function. Depending on therequirements for chemical synthesis, the derivatization can be donebefore entrapment or afterward, using suitable protection anddeprotection methods as needed.

[0135] Since cyclodextrins are composed of carbohydrates, they can besuitably derivatized and coupled through well-known procedures used forother carbohydrates, especially through available hydroxyl groups. Forinstance, vicinal hydroxyl groups on the cyclodextrin can beappropriately oxidized to produce aldehydes.

[0136] In addition, any functional group can be suitably added throughwell-known methods while preserving the cyclodextrin structure andcomplexing properties. Examples are: amidation, esterification,acylation, N-alkylation, allylation, ethynylation, oxidation,halogenation, hydrolysis, reactions with anhydrides, or hydrazines andother amines, including the formation of acetals, aldehydes, amides,imides, carbonyls, esters, isopropylidenes, nitrenes, osazones, oximes,propargyls, sulfonates, sulfonyls, sulfonamides, nitrates, carbonates,metal salts, hydrazones, glycosones, mercaptals, and suitablecombinations of these. The functional groups are then available for thecross-linking of one or more cyclodextrin molecules using a bifunctionalreagent.

[0137] Additional examples of cyclodextrins, inclusion compounds andcatalytic groups including chemical methods for modifying and/orderivatizing cyclodextrins that are useful in the instant invention aredescribed and referenced in U.S. Pat. No. 6,048,736 and PCT/US99/30820,which are incorporated herein by reference.

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[0164] Szejtli, J., et al, Hung. Patent 19,626 (1978)

[0165] Tabushi, I., et al,J. Amer. Chem. Soc. 98/24, 7855-7856 (1976)

[0166] Tabushi I., et al, Tetrahed. Lett. No. 29, 2503-2506 (1977)

[0167] Tabushi, I., Acc. Chem. Res. 15, 66-72 (1982)

[0168] Traut, R. R., et al, Biochem. 12, 3266-3273 (1973)

[0169] Ueno, A., et al, J. Inclus. Phenom. 2, 555-563 (1984)

[0170] VanEtten, R. L., et al, J. Amer. Chem. Soc. 89/13 3242-3253 and3253-3262 (1967)

[0171] Suitable coupling or cross-linking agents for preparing thewater-soluble (or colloidal) CD carriers of the instant invention can bea variety of reagents previously described, including well knowncrosslinkers used to polymerize CD's. Other suitable crosslinkers orderivatizers are various epoxy compounds including propylene oxide,1,2-diethoxyethane, 1,2,7,8-diepoxyoctane, 2,3-epoxy-1-propanol(glycidol), glycerol propoxylate triglycidylether and 1,4-butanedioldiglycidyl ether (e.g. Gramera, or Case, or Johnson, or Parmerter,supra). Also useful are methods employing acrylic esters such asm-nitrophenyl acrylates, and hexamethylenediamine and p-xylylenediaminecomplexes (e.g. Furue, or Harada, or Hatano, or Ogata, supra), andaldehydes, ketones, alkyl halides, acyl halides, silicon halides,isothiocyanates, and epoxides (e.g. Buckler, supra).

Methods for Derivatizing Cyclodextrins

[0172] For this invention, individual cyclodextrin derivatives (i.e.monomer) as well as dimers, trimers and polymers are the primarycomponents, or units used to synthesize the water-soluble (or colloidal)cyclodextrin polymer carriers. Although native cyclodextrins are usefulfor synthesizing the carriers, many other useful properties can beincorporated into the carriers by first derivatizing the cyclodextrincomponents before making the polymers. Derivatizing is defined as thechemical modification of a CD through addition of any functional orcoupling group and/or other substance. Generally, derivatizedcyclodextrins can be used to facilitate cross-linking reactions andintroduce functional groups for use during or after the carrier isprepared. Frequently, an integral part of using derivatizedcyclodextrins involves protecting certain functional groups duringcertain cross-linking steps and then deprotecting those groups for usein subsequent steps.

[0173] A. Protected Hydroxyl Groups.

[0174] Primary and/or secondary hydroxyl groups on the cyclodextrin (orderivatives), can be selectively protected and deprotected using knownmethods during derivatizing and/or capping procedures, to provideselective coupling at the primary or secondary end of the CD molecule,as desired. For instance, formation of protective esters (e.g. benzoatesusing benzoyl chloride), and selective cleavage (deprotection), ofprimary esters using anhydrous alcoholysis (e.g. Boyer, supra), providesmostly primary hydroxyls for derivatization. After derivatization and/orcoupling the primary hydroxyls, the secondary hydroxyls can bedeprotected for additional derivatization, coupling and/or capping.

[0175] Preferred hydroxyl protection schemes include various methods forsilylation of the primary hydroxyls using tert-butyldimethylsilylchloride (TBDMS), (K. Takeo, et al., Carbohydrate Res. 187, 203 (1989))for derivatization of the secondary hydroxyls. Or, the use of sodiumhydride with TBDMS (S. Tian, et al., Tetrahedron Lett. 35, 9339 (1994))to protect secondary hydroxyls during derivatization of the primaryhydroxyls. The silyl groups are then removed by treatment withtert-butylammonium fluoride.

[0176] B. Preparation of Sulfonylated Cyclodextrin

[0177] A variety of suitable methods are available for sulfonylation ofCD or CD polymer before or after protection of specific hydroxyl groups(e.g. Bergeron, Boger or Ueno, supra), and/or capping of the CD (e.g.Emert or Tabushi, supra). Suitably, CD polymer (10 gm), is combined witha suitable sulfonylating reagent (20 gm), such as p-toluenesulfonyl(tosyl) chloride, mesitylenesulfonyl chloride or naphthalenesulfonylchloride, among others, in anhydrous pyridine, for 3-5 Hrs at roomtemperature (RT).

[0178] C. Preparation of Oxidized Cyclodextrin for DialdehydeCyclodextrin (Dial-CD).

[0179] A dialdehyde CD derivative (dial-CD) and dialdehyde cyclodextrinpolymer (dial-CD polymer) is prepared from oxidized cyclodextrin oroxidized CD polymer by oxidation using known methods (e.g. Royer orKobayashii, supra), with sodium metaperiodate in water or suitablebuffer solution (e.g. 0.2 M phosphate saline, pH 5-7), where one or moredialdehydes can be produced per CD. For use in preparing cyclodextrinpolymer carriers, dial-CD can also include oxidized forms of HPCD, DHPCDand SBE-CD.

[0180] D. Amino-Cyclodextrin (Amino-CD) Derivatives.

[0181] Amino groups can be introduced into CD polymer by reaction of asulfonylated CD polymer with azide compounds including hydrazine, and2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone (e.g. Ikeda, supra),or coupling to diamines as described by Kawaguchi, or Matsui, supra.

[0182] Also, when desired, a “monoamino” CD, wherein one amino group hasbeen coupled, can be prepared through known methods, including limitedor sterically determined monosulfonylation, and/or by specificprotection and deprotection schemes. An amino-CD or amino-CD polymer, issuitably protected and/or deprotected as needed.

[0183] E. Diamino Derivatives.

[0184] A previously sulfonylated CD or CD polymer is suitably iodinatedso that it will couple to primary amino groups, using known methods(e.g. Ikeda or Iwakura, supra). Suitably, 10 gm of sulfonylated CD or CDpolymer is combined with 12 gm of NaI on 200 ml of methanol, and mix at70° C. for 48-60 Hrs. The iodinated CD product is collected byprecipitation with acetone and purified by column chromatography.

[0185] The iodinated CD or CD polymer is coupled through an amino groupto a suitable diamino substance. Suitable diamino substances are;1,4diaminobutane, 1,6-diaminohexane, 1,7-diaminoheptane,1,8-diaminooctane, 1,10-diaminodecane, 1,12-diaminododecane, and otheraliphatic, or aromatic, or heterocyclic carboxylic acids with twoavailable amino groups for coupling. Coupling is done in a suitablesolvent such as dimethylformamide (DMF), mixing 10 gm of iodinated CDpolymer with a molar excess of the diamino substance (e.g. 10-20 gm of1,6-diaminohexane), at 100° C. for 24 Hrs. The product, amino-CD (oramino-CD polymer), is concentrated and purified by columnchromatography.

[0186] F. Protected Amino Groups.

[0187] The amino groups introduced by various methods can be suitablyprotected by reaction with a halogenated alkylphthalimide such asN-(4-bromobutyl)phthalimide. After other suitable derivatizing, couplingand/or capping has been done, an amino group is deprotected by reactionwith hydrazine in suitable solvent.

[0188] Also, the diamino substances of various chain lengths can besuitably derivatized before coupling. For instance, they can be “halfprotected” as trifluoroacetamidoalkanes at one of the amino ends, asdescribed by Guilford, H., et al, Biochem. Soc. Trans. 3, 438 (1975),before coupling, and then suitably deprotected such as by hydrolysis oralcoholysis. Yet another suitable method involves the coupling ofTHP-protected amnino-alkynes, previously described, to the iodinated CDor CD polymer and subsequent deprotection as needed.

[0189] G. Sulfhydryl-Cyclodextrin (SH-CD) Derivatives.

[0190] A sulfhydryl group can be added to an amino-CD, suitably preparedas described previously, by coupling the appropriate thiolating agent tothe available amino group. For instance, thiolation of amino groups onamino-CD can be done by known methods using S-acetylmercaptosuccinicanhydride (SAMSA), (e.g. Kiotz, Rector, or Lui supra), SIAB, or2-iminothiolane (e.g. Traut, supra). The sulfhydryl is protected as adisulfide during subsequent coupling reactions until it is exposedthrough disulfide cleavage.

[0191] Sulfhydryls can also be introduced through reaction of availablehydroxyls with a suitable epoxy compound. For instance, epichlorohydrinor a suitable diepoxy crosslinker previously described, is coupled to aCD or CD polymer wherein free epoxy groups are produced. Free epoxygroups are then reacted with sodium thiosulfate to give thiosulfateesters (e.g. Carlsson, supra). The thiosulfate esters are subsequentlyreduced to sulfhydryls with dithiothreitol.

[0192] H. Cyclodextrin Dimer, Trimer and Polymer Derivatives.

[0193] Certain CD dimers, CD trimers and small CD polymers have beenderivatized and can function as carriers or excipients without furthercrosslinking. Sulfate groups can be introduced by reacting primary orsecondary hydroxyl groups with various cyclic sultone compounds toproduce sulfoalkyl ether derivatives. For instance, 1,4-butane sultonereacts with the hydroxyl groups to produce a sulfobutyl ether (SBE)group (Stella, et al. U.S. Pat. No. 5,134,127), or 1,3-propane sultonereacts with the hydroxyl groups to produce a sulfopropyl ether group(Szejtli, supra).

[0194] New, more useful excipients with higher binding affinities can beprepared from CD dimers, trimers or polymers than derivatives of singleCD molecules. These new excipients are synthesized by first preparing CDdimers, trimers or polymers of cyclodextrin by crosslinking monomercyclodextrins by various means. For instance crosslinking is done usingbifunctional or multifunctional epoxy crosslinkers such asepichlorohydrin, 2,3 epoxy-1,4-butanedione, glycerol diglycidyl ether,or glycerol propoxylate triglycidyl ether, among others.

[0195] Then the crosslinked CD products are derivatized with a cyclicsultone such as 1,4-butane to provide sulfobutyl groups or 1,3-propanesultone to provide sulfopropyl groups in basic conditions such as 1-50%NaOH in water.

[0196] Also, such CD dimers, CD trimers and small CD polymers can bederivatized to provide hydroxyethyl, hydroxypropyl or dihydroxy propylgroups by derivatizing them with ethylene oxide, propylene oxide orglycidol. Other useful derivatives include CD dimers, trimers orpolymers with carboxylate groups using methods disclosed or referencedherein.

[0197] Also, phosphate groups can be added to CD dimers, trimers orpolymers by several known methods. For example, E. Tarelli, et al.,Carbohydrate Res. 302(1-2), 27-34 (1997) describes reactingcyclodextrins with inorganic metaphosphates in aqueous solution at pH 4,drying and warming to produce monophosphate esters.

[0198] Other useful derivatives include CD dimers, trimers or polymersthat have been oxidized, such as with NaIO_(4,) to produce dialdehydegroups. The dialdehydes are then coupled to any suitableamino-containing or sulfhydryl-containing compound to provide thedesired derivative.

[0199] The resulting derivatives are generally more soluble that theinitial crosslinked CD dimers, CD trimers and CD polymers and would besuitable for use as drug or other active agent carriers or asexcipients. They are usually more ionic to allow migration in anelectric field for applications such as iontophoresis.

[0200] I. Preparation of Carboxylic Acid CD Derivatives.

[0201] A preferred method for adding carboxylate groups is to coupleglutaric or succinic anhydride to a hydroxyl group on the CD, or CDdimer, trimer or polymer. This produces a terminal carboxylate, whichcan then be protected by esterification as needed. Also, carboxylatescan be derivatized to an NHS ester using N-hydroxysuccinimide andcarbodiimide such as dicyclohexyl carbodiimide.

[0202] Alternatively, a previously sulfonylated CD or CD polymer can besuitably iodinated as previously described for diamino groups. Aniodinated CD polymer or a dial-CD polymer is coupled through the aminogroup to a suitable amino-carboxylic acid to provide the desired lengthof spacer. Suitable amino-carboxylic acids are; 4-aminobutyric acid,6-aminohexanoic acid, 7-amninoheptanoic acid, 8-aminocaprylic acid,12-aminododecanoic acid, and other aliphatic, or aromatic, orheterocyclic carboxylic acids with an available amino group forcoupling.

[0203] Coupling of amino-carboxylic acid to iodinated CD or CD polymeris done in a suitable solvent such as dimethylformamide (DMF), mixing 10gm of iodinated CD polymer with a molar excess of amino-carboxylic acid(e.g. 10 gm of 6-aminohexanoic acid), at 100° C. for 24 Hrs. Theproduct, CD-carboxylic acid, is concentrated and purified by columnchromatography.

[0204] Coupling of amino-carboxylic acid to dial-CD or dial-CD polymeris done by reductive alkylation. In a suitable buffer (e.g. 0.1 Mborate, pH 7.5-8.5, 0.1-0.5 M triethanolamine), 10 gm of dial CD polymeris mixed with a molar excess of amino-carboxylic acid (e.g. 10 gm of12-aminodecanoic acid), at RT for 1-2 Hrs. The Schiffs base coupling isstabilized by suitable reduction with NaBH₄ (e.g. 0.1-1 mg/ml), for 1-12Hrs. The product, CD-carboxylic acid, is concentrated and purified bycolumn chromatography and dried for subsequent reactions as needed.

[0205] J. Capping Cyclodextrins.

[0206] Capping is a type of derivatizing defined herein as coupling anysuitable chemical “capping substance” to two or more sites on the CDmolecule so that the substance spans the area between the coupled sites.Preferably, the capping substance spans across one of the end openingsof the CD molecule and thereby stops the passage of a guest moleculethrough the capped CD molecule.

[0207] It is well known that capping with disulfonyl chloride compoundsis also useful for synthesizing bifunctional derivatives ofcyclodextrins. For instance, when the CD has been capped with a suitabledisulfonyl compound, it is coupled at two of the available hydroxylgroups. These two coupled sites can then be disubstituted to introducevarious thiol or amino groups through nucleophilic displacement(Tabushi, supra). For instance, displacement with ammonia gives aminogroups, displacement with hydrogen sulfide gives thiol groups.

[0208] The CD's used herein can be suitably complexed with one or moreguest molecules and/or derivatized and/or capped before, during or aftertheir incorporation into the water-soluble CD polymer carrier of theinstant invention. In addition, the derivatizing and/or capping can be adone to produce CD's with the desired substances coupled to specificlocations on the CD molecule. In the preparation of CD derivatives foruse as hosts for drugs or other agents, modifications that increaseaffinity between the host CD and guest(s) are preferred. For instance,the host CD's of this invention are preferably derivatized (e.g.methylated or benzylated), and/or capped by various means to increasehost-guest affinity.

[0209] K. Derivatizing and Capping Substances.

[0210] Preferably, the capping substance is coupled at the primary orsecondary “end” of the CD molecule, forming a bridge across either (orboth) opening(s) that includes suitable hydrophobic groups in thecapping substance. The capping substances can be coupled directly toavailable hydroxyls on the CD, or they can be coupled to suitablefunctional groups such as; diamino (or triamino), compounds to iodinatedCD, or azido compounds to sulfonylated hydroxyls, and/or through“spacers” added to the CD.

[0211] Suitable disulfonyl capping substances arebiphenyl-4,4′-disulfonyl chloride, 1,3-benzene disulfonyl chloride,2,4-mesitylene disulfonyl chloride, 2,6-naphthalene disulfonyl chloride,4,4′-oxybis(benzene sulfonyl chloride), 4,4′-methylene bis(benzenesulfonyl chloride), m,m′-benzophenone-disulfonyl chloride,p,p′-stilbene-disulfonyl chloride, and diphenylmethane-p,p′-disulfonylchloride, among others. Other suitable capping substances areimidazoles, 6-methylamino-deoxy and 6-methylamino-6-deoxy derivativestransformed to the corresponding N-formyl compounds, terephthaloylchloride, dianhydrides such as 3,3′,4,4′-benzophenonetetracarboxylicdianhydride and 3,4,9,10-perylenetetracarboxylic anhydride, azidocompounds such as 2,6-bis(4-azidobenzylidene)-4methylcyclohexanone, andderivatives of aurintricarboxylic acid (e.g. thionyl chloridederivatives, triammonium salts “aluminons”), among others (e.g. Szejtli,Emert, Tabushi, or Cramer, supra).

Preparation I Cyclodextrin Polymer Carrier with Completely EntrappedAnthracene

[0212] The purpose is to prepare a water-soluble (or colloidal)cyclodextrin polymer carrier with completely entrapped anthracene. Inthis preparation, beta cyclodextrin was cross-linked while complexedwith anthracene at a molar ratio of 4:1. The procedure was to combine 10ml of water containing 0.0002 moles of cyclodextrin with 1 ml ofchloroform containing 0.00005 moles of anthracene. After about 15minutes of mixing at about 20,000 rpm with a stainless steel impellerand Dremel motor, most of the solvent had evaporated. While stillmixing, 0.4 ml of epichlorohydrin and 0.2 ml of 2 N NaOH was added.After about 20 minutes, the reaction was stopped by adding 0.4 ml ofethanolamine. The resulting solution was allowed to settle and examinedover UV illumination.

[0213] The turbid solution had a greenish-yellow, fluorescent top layerindicating unincorporated anthracene. However, the aqueous phase of thesolution showed a distinct blue fluorescence, indicating that someanthracene was complexed in the cross-linked cyclodextrin polymersuspended in the aqueous phase.

[0214] The preparation in the aqueous phase was separated andconcentrated by evaporation and then extracted 3 times with 4 ml offresh chloroform by mixing, settling and drawing off the solvent phase.The preparation was resuspended in water and produced a turbidsuspension that was still blue fluorescent. Since chloroform extractiondid not remove the anthracene, it showed that the anthracene wascompletely entrapped within the cyclodextrin polymer.

Preparation II Cyclodextrin Polymer Carrier with Completely Entrapped2AA

[0215] The purpose is to prepare a water-soluble (or colloidal)cyclodextrin polymer carrier with completely entrapped 2-aminoanthracene(2AA). The procedure was to combine 0.5 ml of 4.4% beta cyclodextrin inwater, with 0.02 ml of solution containing 80% 1,4 butanediol diglycidylether (BDE), 10% of 0.1 M guest molecule, 2-aminoanthracene indimethylformamide, and 2.4% 2 N NaOH while mixing vigorously andincubating at 60° C.

[0216] After about 1 hour, 0.2 ml of 0.01 M K₂HPO₄ (K2 buffer, pH 8.6),and 0.02 ml more of BDE was added and mixed to continue thecrosslinking. After about one half hour more, the mixture was mixed with0.1 ml of 1M lysine for about 2.5 hours more. The preparation was thencentrifuged for 8 minutes at 2500 rpm and 0.55 ml of supernatant wasfractionated on a column of Sephadex® G-25 (14×0.8 cm) equilibrated withK2 buffer.

[0217] The 0.5 ml fractions were then collected and examined for colorto indicate the presence of the guest molecule 2AA. Fractions were alsotested for carbohydrate to indicate cyclodextrin polymer. To a 50 μlaliquot of polymer fraction in water was added 1 drop of test reagent (3gm potassium dichromate, 10 ml conc. H₂SO₄ and 290 ml water). Themixture was heated gently to oxidize the samples. The intensity of thedark residue was graded on a scale of 1 to 10.

[0218] The carbohydrate test showed that the polymerized cyclodextrinwas in fractions 4 through 8, which was in the area of the void volumedetermined previously with a blue dextran control sample. Also, yellowcolor was seen in corresponding fractions 4 through 6, showing thatguest molecule 2AA could not be separated from the polymer on thecolumn. The carbohydrate (cyclodextrin) test and yellow color testresults for the column fractions are shown in Table A below (Exper. Nov.14, 1989). TABLE A CD Polymer Fraction 1 2 3 4 5 6 7 8 9 RelativeCarbohydrate 0 0 5 9 10 10 10 9 5 Yellow Color No No No Yes Yes Yes NoNo No

Preparation III Cyclodextrin Polymer Carrier with Tethered Guest

[0219] The purpose is to first prepare a water-soluble (or colloidal)cyclodextrin polymer using 1,4 butanediol diglycidyl ether (BDE) tocrosslink with the cyclodextrin hydroxyl groups. Additional BDEmolecules are allowed to randomly couple at only one end before excesslysine is added. The lysine is covalently incorporated by covalentlycoupling to free ends of the BDE previously coupled to the cyclodextrin.The combination of BDE and lysine functions as a spacer group on thecyclodextrin polymer. The fluorophore 2-aminoanthracene is thencovalently tethered as a captured guest to the cyclodextrin polymerthrough the amino group on the BDElysine spacer using glutaraldehyde.

[0220] A. Preparation of Cyclodextrin Polymer and Incorporated Lysine.

[0221] The procedure was to combine 2 ml of 4.4% beta cyclodextrin inwater, 0.1 ml of 2 N NaOH and 0.116 ml of 1,4 butanediol diglycidylether (BDP) while mixing and incubating at 50° C. The molar ratio of BDEto cyclodextrin was about 5:1. After about 4 hours, a 0.5 ml aliquot ofthe mixture was mixed with 0.2 ml of lysine (0.8 M in water,neutralized) for about 1.5 hours. The CD polymer was then fractionatedon a column of Sephadex® G-25 (21×0.8 cm) equilibrated with distilledH₂O and pre-calibrated with free cyclodextrin.

[0222] The 1 ml fractions were then collected and tested forcarbohydrate to demonstrate cyclodextrin polymer. To a 50 μ1 aliquot ofpolymer fraction in water was added 1 drop of test reagent (3 gmpotassium dichromate, 10 ml conc. H₂SO₄ and 290 ml water). The mixturewas heated gently to oxidize the samples. The intensity of the darkresidue was graded on a scale of 1-10. The polymerized cyclodextrin wasin the fractions (3-4) containing a carbohydrate peak that eluted wellahead of the free cyclodextrin control (which peaked at fraction 9). TheCD polymer fractions (3,4) were pooled.

[0223] B. Preparation of CD Polymer with Tethered Fluorophore (FL-CD).

[0224] The CD polymer with lysine was then coupled through the lysinegroups to the guest molecule 2-aminoanthracene by a two stepglutaraldehyde method based on Guesdon, J-L, et al, J of Histochem.Cytochem. 27, 1131-1139 (1979). The procedure was to combine 0.9 ml ofthe CD polymer with 0.1 ml of 25% glutaraldehyde (in water) and 0.02 ml2 N NaOH (starting pH 12), and mix for about 25 minutes. The mixture wasfractionated to remove excess glutaraldehyde on a column of Sephadex®G-25 (9×0.8 cm) equilibrated with distilled H₂O, collecting 0.3 mlfractions. The polymer fractions were pooled in a 1.4 ml volume. The2-aminoanthracene was then coupled by mixing in a total of 0.06 ml of 5mM 2-aminoanthracene in methanol:chloroform (4:1) and 0.01 ml 2 N NaOH(starting pH 12). This was reacted for 4 hours then blocked with 0.1 mlof ethanolamine. The Schiff base coupling was stabilized by adding 0.01gm of NaBH₄ and incubating overnight.

[0225] The mixture was then neutralized with 1 N HCl and excess2-aminoanthracene was removed by fractionating on a column of Sephadex®G-25 (9×0.8 cm) equilibrated with distilled H₂O, collecting 0.5 mlfractions. The fractions were then tested for carbohydrate as describedpreviously and those with carbohydrate were also tested for guestmolecule using chemiluminescence (CL). The CL procedure was to activatethe 2-aminoanthracene using oxidation of bis(2,4,6-trichlorophenyl)oxalate ester (TCPO). Into an FL-CD sample (0.02 ml in 0.1 ml of 0.1 MK₂HPO₄), was added 0.01 ml 0.22% TCPO in ethyl acetate. After placingthe sample into a dark chamber in the luminometer, 1 ml of 0.4 M H₂O₂was injected and the light emission recorded on a chart recorder. Thecarbohydrate (cyclodextrin) test and CL test results for the fractionsare shown in Table B below Exper. CD/1). TABLE B FL-CD Polymer Fraction1 2 3 4 5 6 7 8 9 Relative 5 7 9 9 10 8 6 5 NT Cyclodextrin Relative .25.30 3.33 10.0 >10 3.6 1.1 .40 .28 CL Emission

[0226] These data show that the carbohydrate peak also corresponds tothe most fluorophore CL activity. This CL activity shows that the guestmolecule 2-aminoanthracene is coupled to the CD polymer and could not beseparated by column chromatography.

Preparation IV Cyclodextrin Polymer Carrier Targeted with AntibodyProtein

[0227] The purpose is to synthesize a targeted cyclodextrin polymercarrier by covalently coupling a biorecognition molecule to acyclodextrin polymer carrier. In this example, the carrier was preparedas in Preparation III, and the biorecognition molecule is antibodyprotein.

[0228] A. Preparation of FL-CD Polymer with CoupledN-Hydroxysuccinimidyl (NHS) Ester.

[0229] In this step a cyclodextrin polymer carrier is covalently coupledwith NHS ester to form a NHS-CD. FL-CD polymer carrier (with tethered2-aminoanthracene) was prepared as above and fractionated by columnchromatography using Sephacryl® S200 in a 1.5×18.5 cm columnequilibrated with water (Exper. CD/8). The purified FL-CD was collectedin 1 ml fractions #8-19, and pooled to give a greenish-yellowfluorescent solution. The solution was dried at 60° C. to give about0.36 gm. The product was dissolved in water and titrated to pH 6 with 6N HCl giving 0.144 gm FL-CD polymer carrier per ml.

[0230] The procedure is to form NHS esters with the carboxylic acidgroups on the lysine that is incorporated into the FLCD polymer carrier.To 1 ml of dissolved FL-CD polymer carrier was added 0.1 gin ofN,N′-dicyclohexylcarbodiimide PCC) and mixed to dissolve. Then 0.1 gm ofN-hydroxysuccinimide was added with mixing. After about 1.5 hours, 0.05ml of glacial acetic acid was added and mixed about 25 minutes. To themixture was added about 4 ml of anhydrous methanol, then it was mixed,centrifuged and the light yellow supernatant was collected. Theresulting solution of FL-CD polymer with coupled NHS ester groups wasconcentrated by evaporation and stored in the refrigerator.

[0231] B. Coupling of Gamma Globulin with the FL-CD Polymer Carrier.

[0232] In this step the purpose is to covalently couple antibody protein(human gamma globulin) to cyclodextrin polymer carrier with tetheredguest 2-aminoanthracene.

[0233] To a glass test tube was added 0.2 ml of 0.1 M K₂PO₄, pH 8.5 inwater, 0.1 ml 1.6% human gamma globulin and 0.2 ml of 50% methanolcontaining about 0.09 gm/ml of FL-CD polymer carrier with coupled NHSester. The pH of the mixture was adjusted to about pH 7 with 2 N NaOHand incubated about 2 days at RT. The labeled protein was recovered byprecipitation by adding 1.5 ml of 52% (NH₄)₂SO₄ in water to the mixtureand centrifuging to collect the precipitate. The precipitate wasdialyzed against distilled water to remove (NH₄)₂SO₄ and concentrated toa final volume of 0.11 ml.

[0234] Aliquots of the targeted cyclodextrin polymer carrier were testedfor CL activity using TCPO as described previously. The peak height ofCL activity of the carrier was low but the CL activity continued for alonger time when compared to the FL-CD polymer carrier alone and tocontrol gamma globulin. The CL activity showed that the gamma globulinbiorecognition molecule was coupled to the FL-CD polymer carrier.

Preparation V Cyclodextrin Polymer Carrier with Completely EntrappedPaclitaxel (Taxol)

[0235] The purpose is to synthesize a water-soluble (or colloidal)cyclodextrin polymer carrier that contains completely entrappedpaclitaxel (PTX) and the polymer includes acid-labile hydrazone linkagesthat provide controlled release. The following Table C is a schematic ofthe reactions employed. TABLE C

Cyclodextrin Hydrazone Linkages Cross-linked Aldehydes with TerminalAmines Polymer

[0236] A. Preparation of Dialdehyde CD Using Oxidation.

[0237] The purpose is to produce oxidized cyclodextrin (CD) to providedialdehydes that can subsequently be reacted with hydrazine to form anacid-labile hydrazone linkage. The hydrazone linkages on eachcyclodextrin will also have terminal amino groups for subsequentcrosslinking to make the polymer carrier.

[0238] The oxidation procedure is based on published methods used tooxidize other polysaccharides and specifically cyclodextrins (Kobayashisupra). This method introduces dialdehyde groups at the C-2,C-3-trans-diol position of the cyclodextrin glucose residues.

[0239] The procedure was to add sodium m-periodate (NaIO₄) to 30 mMcyclodextrn in 100 ml of water while mixing at 30° C. The molar ratio ofNaIO₄ to cyclodextrin was 2:1, to give 1 to 2 dialdehydes per CDmolecule. The reaction was continued in the dark for 6 to 8 hours.Remaining NaIO₄ was consumed with a molar excess of ethylene glycol. Theresulting dialdehyde cyclodextrin (dial-CD) was fractionated using gelfiltration on a Sephadex™ G-25 column. The more open dial-CD moleculeshave been found to elute ahead of the native CD. The fractions wereconcentrated by evaporation under vacuum.

[0240] The amount of CD (mw 1135) as carbohydrate in each fraction ismonitored by a colorimetric test for carbohydrates. To 2 ml of watercontaining diluted dial-CD fraction (0.01-0.05 mg) is added 0.05 ml of80% phenol. Then 5 ml of concentrated sulfuric acid is added rapidly tomix. Color is allowed to develop 20 minutes at 25-30° C. and theabsorbance is read at 490 nm. The absorbance is compared to a series ofidentically treated CD standards at 0.005, 0.01, 0.02, 0.04, 0.08 and0.1 mg per m H₂O.

[0241] B. Preparation of Hydrazone Linkages on the CD.

[0242] This reaction involves a condensation reaction of the hydrazinewith available aldehydes to produce a hydrazone linkage. The objectiveis to react dial-CD with enough hydrazine so that ideally each availablealdehyde is coupled to a single hydrazine with minimal cross-linking.The dial-CD preparation is dissolved in water to give startingconcentrations of 30 mM. While stirring the solution at roomtemperature, a 3 to 4-fold molar excess of hydrazine (Sigma) is addedwith continued stirring for 2 hours. The resulting hydrazonecyclodextrin (Hz-CD) is fractionated on a Sephadex™ G-15 column and thefractions dried to constant weight by vacuum evaporation.

[0243] The number of amino groups is determined colorimetrically using aBlue G-250 assay reagent for protein (Reagent Kit Cat #23200, Pierce,Rockford Ill.) with the absorbance read at 595 nm. To ensure that enoughamino groups are available, the Hz-CD fractions with at least 2 freeamino groups available per mole are used in the next step.

[0244] C. Preparation of CD Polymer with Completely Entrapped Drug.

[0245] The purpose is to cross-link Hz-CD to form a water-soluble (orcolloidal) cyclodextrin polymer carrier that is acid-labile. Thepolymers preferably have molecular weights of 20,000-50,000, althoughhigher or lower molecular weights can be used. In this procedure theHz-CD monomers are cross-linked through the terminal amino groups on thehydrazine derivatives.

[0246] In order to entrap the drug, the paclitaxel (PTX) is dissolved ina solvent and mixed with the Hz-CD to form inclusion complexes. Then theHz-CD is cross-linked to form the polymer and completely entrap the drugin polymer aggregates.

[0247] Preparations can be made with molecular ratios between 1:1 and1:8 of PTX to Hz-CD. A near saturated suspension of Hz-CD is prepared in0.05 M phosphate buffer, pH 7.5 (PB). The FIX (about 2 mM) in methanolis added with vigorous mixing (20,000 rpm impeller). While mixing, thedrug is exposed to the aqueous phase to allow complexes to form betweenthe PTX and Hz-CD. Mixing is continued for 15 minutes to one hour. Thecross-linking reagent is then be added while continuing to mix.

[0248] At this point, a variety of amino-reactive, bifunctionalcleavable or noncleavable agents with different spacer lengths can beused to cross-link amino groups. For this example, cross-linking is donewith a bifunctional cleavable coupling agent Tech. Bull. #0544, PierceChem. Co., Rockford Ill.), dithiobis(succinimidyl propionate) (DSP,spacer length 12 angstroms). During polymerization, the objective is tocompletely entrap the drug in polymer aggregates that are soluble (orcolloidal). The cross-liking reaction is run for about 3 hours or justbefore insoluble polymers form.

[0249] The resulting PTX-loaded CD polymer (PT-CD) is then fractionatedby gel exclusion chromatography on pre-calibrated columns of Sephacryl®S200-HR (40×5 cm) equilibrated with PB. Pre-calibration is done usingvarious molecular weight dextrans (i.e. 15,000 to 60,000, Sigma) inseparate runs.

[0250] The PIX-CD fractions that elute in molecular weight rangesbetween 20,000 and 50,000 are vacuum dried at 50° C. and weighed. Forsome procedures, fractions may be concentrated by centrifugal filtrationusing suitable molecular weight cutoff filter tubes (Micron SeparationsInc., Westboro Mass.). Other fractions of higher or lower molecularweight may also be suitable. The approximate moles of product arecalculated as total grams of dried carrier divided by the apparentmolecular weight.

[0251] Alternatively, suitable derivatives of cyclodextrin can be usedto prepare the carrier including CD-blocks described previously.Preferred CD derivatives are hydroxypropyl cyclodextrin (HPCD) and2,3-dihydroxypropyl cyclodextrin (DHPCD). For oxidation to dialdehyde, apreferred form of HPCD or DHPCD is one with 3-4 degrees of substitutionwith propylene oxide or glycidol (Pitha, supra).

[0252] Alternatively, other cross-linkers that provide longer spacerlengths to avoid steric hindrance can be used. One example is ethyleneglycol bis(succinimidylsuccinate) (EGS, 16.1 angstrom spacer). EGS iscleavable with hydroxylamine

[0253] Also, the Hz-CD can be polymerized using the water-solublebifunctional reagent dimethyl adipimate (DMA, 8.6 angstrom spacer,Technical Bull #0438, Pierce). The parameters of molar ratios andreaction times for cross-linking Hz-CD with DMA are suitably optimizedfor the desired polymer size. A near saturated suspension of Hz-CD andPTX in methanol is prepared in PB with vigorous mixing as describedpreviously. As the solvent evaporates, the drug is forced into theaqueous phase to allow complexes to form between the PITX and Hz-CD. TheDMA is added and mixed for 2-6 hours. The resulting PTX-CD isfractionated by gel exclusion chromatography as described previously.The hydrazone linkages provide controlled release when hydrolyzed torelease free drug maximally at pH 4-5.

Preparation VI Targeted Cyclodextrin Polymer Carrier with EntrappedDoxorubicin

[0254] A. Preparation of CD Polymer with Incorporated Amino Groups.

[0255] The purpose is to cross-link gamma cyclodextrin (Mol. Wt. 1297),to form a water-soluble (or colloidal) cyclodextrin polymer carrier thathas completely entrapped doxorubicin (DOX). Cyclodextrins arecrosslinked through their hydroxyl groups to each other using 1,4butanediol diglycidyl ether (BDE, Mol. Wt. 202.2).

[0256] In order to entrap the drug, the DOX is dissolved in a solventand mixed with the CD to form inclusion complexes. Then the CD iscross-linked to form the polymer and completely entrap the drug inpolymer aggregates.

[0257] A preparation is made to give a molecular ratio between 1:2 and1:8 of DOX to CD. While mixing, the drug is exposed to the CD to allowcomplexes to form between the DOX and CD. The procedure is to combinenear saturated DOX with 100 ml of 4.0% cyclodextrin in 0-20% (v/v)dimethylformamide (DMF) in water, with 5 ml of 2 N NaOH (starting pH13), with vigorous mixing (20,000 rpm impeller). The cross-linkingreaction is initiated by adding 10 ml of 95% BDE while ring andincubating at 60° C.

[0258] The reaction is conducted for 2-6 hours followed by the additionof a molar excess of lysine (0.75 ml of 4 M lysine in water, adjusted topH 8). Lysine is incorporated into the polymer as the BDE cross-linksthe lysine through one of its amino groups to the cyclodextrin. Theexcess lysine also couples to and blocks any remaining free BDE. Mixingis continued for one more hour and the mixture is then neutralized with1 N HCl.

[0259] Aliquots of the drug-loaded CD polymer carrier are thenfractionated by gel exclusion chromatography on pre-calibrated columnsof Sephacryl® S200-HR (40×5 cm) equilibrated with distilled H₂O.Pre-calibration is done using various molecular weight dextrans (i.e.15,000 to 60,000, Sigma) in separate runs.

[0260] The carrier fractions that elute in molecular weight rangesbetween 20,000 and 50,000 are taken to the next step. Other fractions ofhigher or lower molecular weight may also be suitable. The fractions arethen vacuum dried at 50° C. and weighed. The approximate moles ofproduct are calculated as total grams of dried carrier divided by theapparent molecular weight. The relative amount of CD in the fractionscan be monitored by a calorimetric test as described previously. The CDpolymer fractions can also be tested for the presence of amino groups asdescribed previously.

[0261] If needed, additional lysine molecules can be added to thePolyCD. The procedure is to again treat the carrier with BDE asdescribed above, but for only 20-30 minutes. Additional amino groups arethen introduced with the excess lysine treatment and the product isfractionated on Sephacryl® as described.

[0262] Alternatively, a selective derivatization procedure is used thattakes advantage of the more reactive primary hydroxyls. The procedure isto first “tosylate” two or more primary hydroxyls on each cyclodextrinand then replace the tosyl groups with amino groups. The cyclodextrinsare then complexed with the DOX and polymerized by cross-linking throughthe aminos using a bifunctional cross-linking agent.

[0263] The tosylation step is done by reacting 12 grams of cyclodextrinwith 9 grams of p-toluenesulfonyl chloride (tosyl chloride) in 100 ml ofanhydrous pyridine solvent. The tosyl chloride is added in 3 gramaliquots over a 36 hour period with constant stirring of the mixture fora total of 48 hours. The reaction is stopped with 20 ml of methanol. Theproduct is precipitated, filtered and washed with 200 ml aliquots ofchloroform, then dried.

[0264] The tosyl groups are substituted for azide by dissolving 1.3 gmof the tosylated cyclodextrin in 100 ml of dimethylformamide (DMF) andadding 1 gm of sodium azide. The mixture is heated with siring to 100°C. for 2 hours and the product is collected from dried supernatant. Theproduct is dissolved in 10 ml of water, precipitated with acetone anddried.

[0265] The azide cyclodextrin is reduced to the amine by dissolving 1 gmin 100 ml of 20% methanol/water containing 0.4 gm of palladium blackcatalyst (Sigma). The mixture is stirred 1 hour under H₂, then filterthrough Celite. The amino-derivatized cyclodextrin is collected bydrying.

[0266] The resulting amino-cyclodextrin (amino-CD), can then becomplexed with DOX and polymerized using any water-soluble bifunctionalreagent such as dimethyl suberimidate (DMS, Mol. Wt 273.2), which isroutinely used to selectively couple amino groups Technical Bull.,Pierce).

[0267] The parameters of molar ratios and reaction times forcross-linking amino-CD with DMS are optimized for the desired polymercarrier. Typically, to 100 ml of 4.0% amino-CD previously complexed withDOX in 0.2 M triethanolamine HCl, pH 8.5 in water, is added 4 gm of DMSand me at 60° C. for 2-6 hours. The reaction is stopped by the additionof a molar excess of lysine (4 M, adjusted to pH 8). Lysine isincorporated into the polymer as the DMS cross-links the lysine andadditional amino groups are available for coupling to antibody. Theexcess lysine also couples to and blocks any remaining free DMS. Themixing is continued for one more hour and the mixture is thenneutralized with 1 N HCl. The resulting polymer is fractionated by gelexclusion chromatography as described previously. Alternatively, anysuitable biorecognition molecule with an available amino group can beused in place of the lysine such as antibodies or other proteins,polypeptides, or amino-sugars. Also, other anticancer drugs can be usedin place of DOX such as daunomycin, puromycin or ellipticine.

[0268] B. Introduction of Sulfhydryl Groups by Thiolating Amines on theCD Carrier.

[0269] The CD polymer carrier is thiolated by modifying the lysineresidues using 2-iminothiolane (FW 137.6), based on the technicalbulletin from Pierce Chem. Co. The number of available amino groups onthe carrier can be determined as described previously. The molar ratiothat is used between the carrier and 2-iminothiolane is about 1:10.

[0270] The reaction is carried out by combining 0.4 mmoles of carrierdissolved in 0.16 M borate buffer (pH 8.0), and 4 mmoles of2-iminothiolane. The mixture is mixed for about 2 hours at roomtemperature. The resulting thiolated carrier is separated by gelchromatography using a Sephadex® G15 column equilibrated with 0.05 Mphosphate buffer, pH 7.5.

[0271] Aliquots of the thiolated carrier can be tested for the presenceof sulfhydryl groups. The test for sulfhydryl is a standard test thatemploys 5,5′-dithiobis(2-nitrobenzoic acid, DTNB). The procedure is tocombine 0.1 ml of thio-polyCD samples diluted in water with 1 ml ofdeoxygenated 0.2 M Tris buffer, pH 8.2, and add 0.1 ml of 0.01 M DTNB indeoxygenated methanol. Color is allowed to develop for 30 minutes andthe absorbance is read at 412 nm on a spectrophotometer. The results arecompared to a standard curve of identically tested dilutions of2-mercaptoethanol. The goal is to introduce an average of at least threesulfhydryl groups (2-mercaptoethanol molar equivalents), for each moleof thiolated carrier.

[0272] C. Conjugation of the Thiolated CD Carrier to Maleimide-ActivatedAntibody.

[0273] The molar ratio of thiolated carrier to antibody is about 4:1.For instance, 0.02 mmoles of the 20,000 molecular weight fraction ofthiolated carrier is conjugated with 0.005 mmoles of antibody previouslycoupled through amino groups to m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS-antibody) (MW 140,000). Other molar ratios of thiolatedcarrier to antibody can be used during conjugation.

[0274] The conjugation reaction is to combine freshly preparedMBS-antibody with thiolated carrier in 0.05 M phosphate buffer, pH 7.5,and stir for 2 hours at room temperature. The conjugate is fractionatedby gel exclusion chromatography using bovine IgG calibrated Sephacryl®S200-HR column equilibrated with the same buffer. The fractionatedconjugate is collected in a fraction collector equipped with anultraviolet monitor set at 280 nm to detect the IgG. The conjugate is inthe fractions containing IgG and corresponding to molecular weightsgreater than 200,000.

[0275] The conjugate fractions can be tested for protein content usingthe Bradford calorimetric method and tested for carbohydrate asdescribed previously. Fractions greater than 200,000 molecular weightare pooled and concentrated by centrifugation filtration using 100,000molecular weight cutoff filter tubes (Micron Separations Inc., WestboroMass.) or by precipitation with ammonium sulfate and dialysis againstPB.

Preparation VII Oxidized Cyclodextrin Polymer Carrier with EntrappedDoxorubicin

[0276] (CD41B) The purpose is to prepare oxidize cyclodextrin (CD) toprovide dialdehydes that are then reacted with a diamino compound suchas 1,6 hexanediamine to provide terminal amino groups for subsequentcrosslinking to make the polymer carrier. Alternatively, CD monomers,dimers or polymers can be derivatized using methods previously describedfor introducing active groups for coupling with amino groups. Forinstance, the CD (or CD-block) can be suitably tosylated, or treatedwith various bifunctional epoxy compounds such as BDE or GDE beforecoupling to the amino compound.

[0277] (CD52) In some preparations, CD-block dimers, trimers andpolymers were first synthesized by crosslinking the CD with epoxycoupling agents such as BDE, GDE or TMTE. For GDE, the procedure was tocombine about 4.5 gm of CD dissolved in about 60 ml of 60° C. water withabout 0.1 ml of 10 N KOH and about 0.5 ml of GDE. The solution is mixedvigorously at 60° C. for 2-4 hours or until the GDE is consumed. At thispoint the mixture can be oxidized to produce dial-CD blocks, or theCD-blocks can be treated with glycidol before oxidation to produce“polyaldehyde-CD” for use in the next step. Alternatively, theun-oxidized CD-blocks can be derivatized with amino or thiol groups forsubsequent crosslinking in CD carrier synthesis.

[0278] A. Preparation of Dialdehyde CD Using Oxidation.

[0279] The oxidation procedure is similar to that of Preparation V. Theprocedure was to add 3.42 gm of sodium m-periodate (NaIO₄) to 4.546 gmof beta cyclodextrin dissolved in 60 ml of water and 0.5 ml of 10 N KOHat 70-80° C. The molar ratio of NaIO₄ to cyclodextrin was 4:1. After onehour, the pH of the solution was adjusted up to 14 with 0.03 ml of 10 NKOH and the reaction was continued in the dark overnight.

[0280] The pH was adjusted to 7 with 0.02 ml of 10 N KOH and theremaining NaIO₄ was consumed with a 2× molar excess of ethylene glycol(0.5 ml). The resulting dialdehyde cyclodextrin (dial-CD) was clarifiedby filtration through a 0.2 micron filter and fractionated on aSephadex™ G-25 column. The leading fractions containing dial-CD werepooled.

[0281] In some preparations, the dial-CD was subsequently crosslinkedusing various epoxides such as BDE or GDE, which produced CD-blockdimers, trimers and polymers containing aldehydes. Alternatively, the CD(or CD-block) has been treated with glycidol (2-7× molar excess, pH7-10) before oxidation, which produces 2,3-dihydroxypropyl cyclodextrin(DHPCD) with additional diols at the primary and secondary sides of themolecule. Subsequent oxidation then converts the diols to aldehydes toproduce a “polyaldehyde-CD”, which can then be coupled with aminogroups.

[0282] B. Preparation of 1,6 Hexanediamine-Coupled CD Monomer.

[0283] This reaction involves the reaction of the amino groups on 1,6hexanediamine (HXDA) with available aldehydes on the dial-CD (orpoly-CD) to form aldehyde bonds with amino groups so that ideally eachaldehyde is coupled to a single HFA molecule with minimal cross-linking.About a 4× molar excess of HXDA was added to about 0.0014 moles of thedial-CD preparation in water while stirring at room temperature. The pHwas adjusted to 7 with about 1.3 ml of 6 N HCl, and reacted overnight.The resulting 1,6 hexanediamine-coupled cyclodextrin monomer (HXDA-CD)was concentrated by evaporation to about 15 ml. The entire solution wasapplied to a Sephadex™ G-25 column in water. The fractions were testedcolorimetrically for carbohydrate content and amino groups. The frontfractions containing carbohydrate and amino groups were pooled andHXDA-CD monomer was concentrated by evaporation to about 20 ml.

[0284] Alternatively, other amino compounds have been coupled to theoxidized CD such as hydrazine, adipic add dihydrazide, glutamic acid,beta-phenylethylamine, laurylamine and cystamine. Many other usefulamino compounds can be coupled to the oxidized CD or CD-block such aspolypeptides, 6-amino-N-hexanoic acid, arginine, protamines,N-(2-aminoethyl)-1,3-propanediamine (AEPD), polyethylenimine (PEI) andnucleic acids.

[0285] C. Preparation of HXDA-CD Polymer with Completely Entrapped DOX.

[0286] (CD49) The purpose is to cross-link HXDA-CD monomer through theterminal amino groups to form a water-soluble (or colloidal)cyclodextrin polymer carrier. The cattier is prepared by combiningHXDA-CD monomer with DOX, then adding an “activated” HXDA-CD monomerthat polymerizes with the HXDA-CD and entraps the DOX in situ. The“activated” monomer can be any CD monomer (or CD-block) that has beentreated to provide active coupling groups that will crosslink with theamino groups on the other CD monomer or CD-block. Monomers can beactivated by treating them with a variety of bifunctional couplingagents listed previously. Also, dial-CD can be used as the activatedmonomer where the aldehyde groups can couple to the amino groups of theHXDA-CD.

[0287] In this case, the HXDA-CD is treated with glutaraldehyde to formaldehyde bonds with amino groups before combining it with the othermonomer and drug. Activated monomer was prepared by combining about0.126 gm of HXDA-CD in 2.1 ml water with 0.35 ml of 1 N NaOH, and thenadding 0.35 ml of 20% glutaraldehyde in water. Drug-complexed monomerwas prepared by combining about 0.021 gm of H A-CD in 0.35 ml of waterwith about 3.25 mg of DOX in 0.28 ml of water.

[0288] After about 20 minutes, drug-loaded carrier was prepared bycombining one half of the activated monomer (0.063 gm in 1.4 ml) withthe drug-complexed monomer preparation. A drug-free control carrier wasalso prepared by combining 1.4 ml of the same activated monomer solutionwith 0.35 ml of water containing 0.021 gm of untreated monomer only.Both mixtures were allowed to crosslink for about 1.5 hours, thendialyzed for about 1 hour in 12,400 molecular weight cutoff (MWCO),cellulose tubing against 70% isopropanol in water. Dialysis wascontinued for about three more hours against distilled water.

[0289] After no detectable DOX was found in the dialysate, thepreparations were analyzed for DOX. DOX was measured by diluting 0.02 mlaliquots of sample into 0.18 ml of 1 N NaOH, reading the absorbance at620 nm and comparing the absorbances to a standard curve of DOX.Aliquots of the preparations were also dried to determine the weight %DOX.

[0290] The preparations were then tested in a cytotoxicity assay usingDaudi cells from human Burkitt lymphoma. The test is a colorimetricassay based on the ability of metabolically active cells to reducethiazolyl blue (MTT) to a blue formazan product (Alley, et al., CancerRes. 48:589 (1988)).

[0291] The procedure was to incubate growth phase cells in RPMI mediawith 10% fetal bovine serum and containing different concentrations ofeach carrier preparation for 48 hours. Cytotoxicity was determined as afunction of the concentration of carrier needed to inhibit growth by50%. This was measured by a reduction in the amount of colored productcompared to untreated control cells. With the control cell value takenas zero cytotoxicity, there was a five-fold increase in cytotoxicity ofthe DOX-loaded carrier vs. the control after 48 hours. Results arepresented in the following Table D. TABLE D Weight % Preparation DOXConc. Total Dry Wt. % DOX Cytotoxicity DOX-loaded 1.80 mg/ml 25.0 mg/ml7.2 60.5 Control Carrier 0 27.0 mg/ml 0 10.0

Preparation VIII Beta CD Polymer Carrier Crosslinked Through Sulfhydrylswith Entrapped Doxorubicin

[0292] (CD45) The purpose is to crosslink thiolated cyclodextrinmonomers through their sulfhydryl groups while they are complexed withdrug to completely entrap the drug in the cattier.

[0293] A. Preparation of Thiolated CD Monomer.

[0294] (CD41b) HXDA-CD was prepared as described previously. The HXDA-CDwas thiolated by slowly combining and mixing about 0.6 gm of HXDA-CD in10 ml of 0.1 M K₂HPO₄ buffer, pH 8.5, in water with 0.33 gm ofiminothiolane (2-IT). After about 2 hours, the mixture was fractionatedon a Sephadex™ G25 column in water. The leading fractions containing thecarbohydrate peak were pooled and concentrated by evaporation to giveabout 0.076 gm per ml.

[0295] B. DOX-Loaded Carrier Prepared by Dithiol Crosslinking ofThiolated CD Monomer.

[0296] In duplicate preparations, a 0.2 ml aliquot of the thiolated CDmonomer (about 0.012 mmoles) was combined with about 0.0002 mmoles ofdoxorubicin and mixed to allow complexing. Crosslinking through dithiollinkages was initiated by the addition of 0.05 ml of 30% H₂O₂ andheating over boiling water. Through oxidation and coupling of thesulfhydryl groups to form dithiols, the monomer becomes crosslinked. Apolymer carrier formed that entrapped the DOX in the carrier and formeda red particulate suspension that was precipitated by centrifugation.The carrier preparations were resuspended and dialyzed overnight in12,400 MWCO cellulose tubing against 10% isopropanol. The recovereddialysates were again centrifuged and produced red pellets indicatingthat DOX was still entrapped in the carrier.

Preparation IX Coupling Methods for Targeting Cyclodextrin PolymerCarriers

[0297] These are methods for synthesizing cyclodextrin polymer carrierswherein a coupling group is included in the composition to provide forcoupling to any suitable biorecognition molecule with a suitablefunctional group. The biorecognition molecule can be a suitable protein,including antibodies and avidins or streptavidin, or ligands, or nucleicacids.

[0298] A. Preparation of NHS-CD Polymer Carriers.

[0299] In a suitable anhydrous solvent such as DMF, the CD-carboxylicacid polymer is combined with N-hydroxysuccinimide and an aromaticcarbodiimide such as N,N-dicyclohexylcarbodiimide, at approximatelyequimolar ratios and reacted at RT for 1-3 Hrs. The product,N-hydroxysuccinimide cyclodextrin (NHS-CD), is separated in the filtratefrom precipitated dicyclohexylurea, collected by evaporation andpurified by chromatography.

[0300] Under appropriate conditions, NHS-CD polymer derivatives can beprepared by coupling NHS esters directly to amino-CD or amino-CDpolymer. Preferably, the NHS ester is a bifunctional NHS coupling agentwith a suitable spacer. Suitable NHS coupling agents for use in thisinvention have been previously described, including DSS,bis(sulfosuccinimidyl)suberate (BS³), DSP, DTSSP, SPDP, BSOCOES, DSAH,DST, and EGS, among others.

[0301] B. Preparation of Sulfhydryl-CD Polymer Carriers.

[0302] Sulfhydryls on polymer carriers can be used for disulfidecoupling to other available sulfhydryls on the desired biorecognitionmolecule such as antibodies, or avidins, or streptavidin, or ligands, ornucleic acids. If needed, the available sulfhydryls can be introduced bythiolation of the biorecognition molecule before coupling.Alternatively, a sulfhydryl-containing CD polymer carrier is coupled toany maleimide derivative of protein, ligand, nucleic acid or biotin,(e.g. biotin-maleimide) or iodoacetyl derivatives such asN-iodoacetyl-N′biotinylhexylenediamine.

[0303] C. Maleimido-Cyclodextrin Polymer Carriers and Iodo-CyclodextrinPolymer Carriers.

[0304] The maleimido-cyclodextrin polymer carriers (Mal-CD), of thisinvention are suitable for coupling to native or introduced sulfhydrylson the desired biorecognition molecule.

[0305] A maleimido group is added to an amino-CD polymer carriersuitably prepared as described previously, by coupling a suitablehetero-bifunctional coupling agent to the available amino group. Thehetero-bifunctional coupling agent consists of a suitable spacer with amaleimide group at one end and an NHS ester at the other end. Examplesare previously described and include MBS, SMCC, SMPB, SPDP, amongothers. The reaction is carried out so that the NHS ester couples to theavailable amino group on the CD polymer carrier, leaving the maleimidegroup free for subsequent coupling to an available sulfhydryl on abiorecognition molecule.

[0306] Under appropriate conditions, Iodo-Cyclodextrin (Iodo-CD) polymercarriers can be prepared for coupling to sulfhydryl groups. Forinstance, NHS esters of iodoacids can be coupled to the amino-CDpolymers. Suitable iodoacids for use in this invention are iodopropionicacid, iodobutyric acid, iodohexanoic acid, iodohippuric acid,3-iodotyrosine, among others. Before coupling to the amino-CD polymer,the appropriate Iodo-NHS ester is prepared by known methods (e.g.Rector, supra). For instance, equinolar amounts of iodopropionic acidand N-hydroxysuccinimide are mixed, with suitable carbodiimide, inanhydrous dioxane at RT for 1-2 Hrs, the precipitate removed byfiltration, and the NHS iodopropionic acid ester is collected in thefiltrate. The NHS iodopropionic acid ester is then coupled to theamino-CD polymer carrier.

Preparation X Biotinylated Cyclodextrin Polymer Carriers

[0307] Biotinylated CD polymer carriers can be produced by a variety ofknown biotinylation methods suitably modified for use with CD's. Forinstance, combining an amino-CD polymer derivative with a knownN-hydroxysuccinimide derivative of biotin in appropriate buffer such as0.1 M phosphate, pH 8.0, reacting for up to 1 hour at room temperature.Examples of biotin derivatives that can be used are,biotin-N-hydroxysuccinimide, biotinamidocaproate N-hydroxysuccinimideester or sulfosuccinimidyl 2-(biotinamino)ethyl-1,3′-dithiopropionate,among others.

[0308] Through the use of suitable protection and deprotection schemes,as needed, any CD polymer carrier of the instant invention can becoupled to biotin or a suitable derivative thereof, through any suitablecoupling group. For instance, biocytin can be coupled through anavailable amino group to any NHS-CD label described herein. Likewise,thiolated biotin can be coupled to any mal-CD polymer carrier.

[0309] The biotinylated CD polymer carrier can be used to couple aplurality of carriers to an intermediate. For instance, by combiningdilute solutions of the biotinylated CD carrier with avidin orstreptavidin in the appropriate molar ratio, 1, 2 or 3 biotinylated CDcarriers will couple to the avidin or streptavidin and produce a complexwith one or more biotin-binding sites still available.

Preparation XI A Cyclodextrin Polymer Carrier Prepared on a SolidSupport

[0310] Another embodiment for a water-soluble (or colloidal)cyclodextrin polymer carrier can be synthesized with a more predictablenumber of CD molecules, giving new advantages of uniform structure andchemical properties. The synthesis method is to couple an initial CDmolecule (or derivative) to a solid support using a cleavable couplingagent. Then additional CD molecules (or CD derivatives with or withoutprotected groups) are attached to the first CD in a controlled,step-wise manner. Alternatively, a suitable intermediate substance (i.e.amino derivatized PEG or HPMA) can be initially coupled to the solidsupport and CD molecules coupled to it. After the desired number of CDmolecules have been linked together to form an open polymer, the carrieris then cleaved from the solid support. The desired drug or other activeagent is then allowed to complex with the polymer. Also, the polymer canbe further cross-linked to close the polymer and completely entrap theactive agent.

[0311] The CD molecules used in this procedure can include tetheredguests and antenna substances, and be suitably derivatized and/or cappedbefore coupling to incorporate other desirable features. However, it ispreferred that each CD molecule (or dimer, or trimer), that is coupled,has a well-defined structure to facilitate the production of CD polymercarriers with uniform and consistent properties.

[0312] A variety of suitable materials, such as those used inchromatography (e.g. Smokova-Keulemansova, supra), can be used for asolid support. The solid support can be in the form of particles, beads,fibers, plates, and tubing walls, and composed of styrenes, acrylamides,silica gels, solid or porous glass, dextrans, and celluloses, amongothers that are suitably derivatized as needed and compatible with thereactions used.

[0313] The initial coupling agent used to couple the initial CD orintermediate substance to the support is preferably one that is readilycleaved when desired. Suitably, the initial coupling agent is abifunctional, amino-reactive reagent such as those with a cleavabledisulfide group, including DTBP, DSP, DTSSP and photoactive couplerslike BASED, SADP, SAND, and SASD. Other suitable initial amino-reactivecoupling agents are periodate cleavable, such as DST and sulfo-DST, orhydroxylamine cleavable at the ethyl ester linkage, such as EGS andsulfo-EGS.

[0314] The coupling agents used to couple subsequent CD's to make anopen polymer are preferably noncleavable, or biocleavable, or cleavableby a different mechanism than the initial coupling agent When couplingthrough amino-derivatized CD molecules, amino-reactive, bifunctionalcoupling agents such as DMA, DMP, DMS, DSS and DSG would be used. Whencoupling through sulfhydryl-derivatized CD molecules,sulfhydryl-reactive, bifunctional coupling agents would be used such asMBS. Diepoxy coupling agents such as BDE can be used to couple throughamino, sulfhydryl or hydroxyl functional groups.

[0315] In another embodiment, when the polymer is cleaved from thesupport after synthesis, it can leave a suitable functional group fortargeting by subsequently coupling a biorecognition molecule to thepolymer. Or, the remaining functional group can be converted to an NHSester by various known means for subsequent coupling to an amino groupon a biorecognition molecule.

CD Polymer Carrier Synthesis

[0316] A suitable method for synthesizing water-soluble (or colloidal)cyclodextrin polymer carriers of the instant invention is as follows;

[0317] 1. A suitable amino-derivatized solid support is prepared. Forinstance, porous glass beads or predried silica gel is amino-derivatizedwith (3-aminopropyl)trimethoxysilane. The solid support is then treatedfor coupling (activated), with a bifunctional, cleavable disulfidecoupling agent, DSP. The uncoupled reagents are removed.

[0318] 2. To the support is added for coupling, an excess of amino-CDderivative, or amino-2, 3 dihydroxypropyl beta cyclodextrin(amino-DHPCD) or amino-derivatized HPMA (amino-HPMA). The uncoupledreagents are removed.

[0319] 3. The initial CD or intermediate substance is then treated foradditional coupling (activated) with a suitable bifunctional couplingagent that will react with the initial CD and subsequently couple toadditional CD molecules. For instance, with amino-DHPCD or amino-HPMA,an amino-reactive agent such as DSS is used. Or, the initial CD orintermediate substance can be treated with a diepoxy such as BDE. Theunreacted coupling agent is then removed.

[0320] 4. An excess of amino-DHPCD molecules (which may include somehalogenated alkylphalimide protected amino groups) are then added tocovalently couple to the initial CD or intermediate substance. Theunreacted amino-DHPCD molecules are then removed.

[0321] 5. The immobilized preparation is then treated again foradditional coupling with a suitable bifunctional coupling agent such asDSS or BDE. The unreacted coupling agent is then removed.

[0322] 6. Another cycle of excess amino-DHPCD molecules are then addedas before to covalently couple to the preparation and unreacted reagentis removed.

[0323] 7. Depending on how large an open polymer is desired, the stepsare repeated of activating the preparation again with coupling agent,removing the unreacted agent, adding excess amino-DHPCD molecules forcoupling and removing the unreacted CD molecules. After the last cycleof DHPCD molecules have been coupled to the preparation, unreactedmolecules are removed.

[0324] 8. The cyclodextrin polymer carrier is then cleaved from thesolid support by reduction of the disulfide bond in the initial couplingagent.

[0325] 9. To the open polymer preparation the desired drug or otheractive agent is added and allowed to complex with the polymer. In thisexample, puromycin is added to allow inclusion complexes to form withthe polymer.

[0326] 10. If desired, the drug-loaded polymer can be furthercross-linked to dose the polymer and completely entrap the active agentIn this example, the amino-reactive coupling agent DSS is used tocross-link the available amino groups. If employed, previously protectedamino groups are made available by a deprotection step before finalcross-linking.

[0327] Other modifications can be included before final cleavage. Forinstance, acid-labile linkages can be incorporated into the finalcross-linking to provide controlled release of entrapped active agent.In one embodiment, vicinal hydroxyls on the DHPCD molecules of the openpolymer can be oxidized to dialdehydes using Na metaperiodate. Thedialdehydes are then coupled to hydrazine to provide acid-labilehydrazone linkages with terminal amino groups.

[0328] The open polymer is then loaded with drug as before and thenclosed by cross-linking the terminal amino groups. The finalcross-linking is done using a bifunctional, amino-reactive couplingagent such as DSS or BDE. Also, the drug-loaded carrier can be treatedwith acetic or succinic anhydride to give carboxylates that areconverted to NHS esters through reaction with carbodiimides andN-hydroxysuccinimide. The carrier can also be targeted by coupling it toa suitable biorecognition molecule.

Preparation XII A Cyclodextrin Polymer Carrier Prepared From aCyclodextrin Monolayer

[0329] Another embodiment for a water-soluble (or colloidal)cyclodextrin polymer carrier can be synthesized wherein the cyclodextrinmonomers are first cross-linked to form an open polymer that is in theform of a sheet or layer of cross-linked cyclodextrin molecules. Thisembodiment can provide new advantages of organized structure andchemical properties.

[0330] In the first step of the synthesis method, CD molecules (or CDderivatives such as HPCD or DHPCD) are positioned on a surface so thattheir primary or secondary ends are facing the surface and their edgesare within coupling distance of each other. Ideally, all of the CDmolecules are oriented in the same direction. One way of accomplishingthis is to prepare a surface (i.e. a solid support or flexible surface)onto which guest molecules have been covalently coupled (i.e. throughspacer groups) so that each guest is available to form an inclusioncomplex with a cyclodextrin. Suitably, guest molecules are used thatforce the cyclodextrin molecules to bind to them in only oneorientation. For instance, if the guest molecules are just big enough,they will form the strongest binding inclusion complex by only enteringthe larger, secondary end of the cyclodextrin molecule and not thesmaller primary end. Examples of the most preferable inclusioncompounds, especially with aromatic compounds, are well known for alphaCD, beta CD and gamma CD. For instance, adamantane and anthracenederivatives bind primarily through the secondary end of beta CD andpyrene derivatives bind primarily through the secondary end of gamma CD.In the following example, 2-aminoanthracene can be replaced with asuitable amino-derivatized adamantane such as 1-aminoadamantane,1-adamantane methyamine, or 1-adamantane carboxamide.

[0331] In this example, 2-aminoanthracene is coupled toamino-derivatized glass beads using a bifunctional NHS coupling agentsuch as DSS. The 2-aminoanthracene is immobilized in very high densityso that many molecules are within one beta CD diameter's distance apart(i.e. about 6 angstroms). Then in suitable solvent or aqueous buffer,beta CD is mixed with the beads to form inclusion complexes with theimmobilized 2-aminoanthracene on the bead surface. The excess CD may beremoved.

[0332] Then the complexed CD molecules are cross-linked with a diepoxysuch as BDE, or a triepoxy such as glycerol propoxylatetriglycidylether, so that every CD molecule is coupled to at least two(preferably 3 or 4) of its neighbor CD molecules. Alternatively,derivatized CD molecules can be used such as amino-CD or amino-HPCD oramino-DHPCD, and then cross-linked using a cleavable, bifunctionalcoupling agent such as DSP, DST or EGS. Also, amino-CD molecules can becross-linked using a suitable biocleavable coupling agent describedherein.

[0333] The resulting open polymer is a sheet or monolayer of CDmolecules (or CD derivatives with or without protected groups). Theresulting CD monolayer is then removed from the immobilized2-aminoanthracene molecules by using a suitable competing solvent orsurfactant to cause dissociation.

[0334] The CD monolayer is then mixed, in suitable solvent or aqueousbuffer, with a drug or other active agent (i.e. paclitaxel) to allow themonolayer to form inclusion complexes with the drug. The drug-loaded CDmonolayer is then further cross-linked to close the polymer andcompletely entrap the drug. The resulting CD monolayer carrier can alsobe targeted by coupling it to a biorecognition molecule. The CDmonolayer carrier can be further derivatized to provide functionalgroups that are then used for coupling to the biorecognition molecule.

Preparation XIII CD Polymer Carriers with Antenna Substances

[0335] A new water-soluble (or colloidal) CD polymer carrier withpotentially greater cytotoxic or catalytic efficiency can be synthesizedby incorporating antenna substances. An antenna substance is defined ascertain light and/or energy collecting substances that transfer theenergy to a catalyst or energy emitter in the carrier. The variousantenna substances of the invention can be conjugated and/ornoncovalently coupled in “close proxnity” so that they willcooperatively participate in an energy transfer reaction resulting inthe emission of energy or a product. The most preferred application isin photodynamic therapy where photoactive agents are used to kill cancercells.

[0336] The antenna substances can be coupled to the carrier in variousways to promote the most efficient cytotoxic or catalytic activity. Forinstance, the antennas can be covalently coupled to the CD derivative orCD polymer, to the guest photoactive agent, or to an intermediatesubstance that is part of the CD carrier. Certain photosynthetic antennasubstances (e.g. chlorophylls, pigments) can also be couplednoncovalently to the CD carrier through binding to certain polypeptides(e.g. from photosynthetic plants, algae and bacteria), which are thencovalently bound to the CD carrier. Examples of photosyntheticsubstances are described by H. Zuber, TIBS 11, 414-419, October, 1986,and J. Deisenhofer, et al, Science 245, 1463-1473 (1989), the contentsof which are incorporated herein by reference.

[0337] Suitable antenna substances are any aliphatic, aromatic orheterocyclic compounds that are capable of collecting light energy orphotons. The most preferred antenna substances for use in photodynamictherapy are those that absorb infrared and far infrared light Examplesinclude carotenoids, folic acids, retinols, retinals, rhodopsins,viologens, chlorophylls, bacteriochlorophylls, phycobiliproteins,phycoerythrins, porphyrins, MCe_(6,) open chain tetrapyrroles (bilins),tryptophan and/or tyrosine-containing substances (e.g. polypeptides),Rose Bengal, fluorophores, scintillators, and various derivatives,analogs and precursors of the antenna substances.

Preparation XIV Cyclodextrin Catalyst Agent

[0338] A cyclodextrin catalyst agent is a new invention defined hereinas a cyclodextrin derivative of an individual cyclodextrin, or dimer,trimer or polymer, wherein the CD derivative host functions as an“artificial enzyme”, and certain guest molecules function as chemicalsubstrates. When the chemical substrate comes in contact with thecyclodextrin catalyst agent under appropriate conditions, it is modifiedto produce a product that is inhibitory or toxic to certain cells,microbes or parasites. In one embodiment, the CD catalyst agent iscoupled to a biorecognition molecule for targeting specific infectedcells, cancer cells, tissues or disease organisms.

[0339] With suitable derivatization, the CD catalyst agents can besynthesized to bind and modify prodrugs into active drugs and modifyother specific substrates. The CD catalyst agent can also catalyzespecific reactions such as generate free radicals, including singlet ortriplet oxygen that directly kills infected cells, cancer cells, ordisease organisms.

[0340] Preferred CD catalyst agents include various photosensitizersubstances, especially those used for singlet and triplet oxygenformation useful for photodynamic therapy (van Lier, J. E. In“Photodynamic Therapy of Neoplastic Disease”; Kessel, D., Ed., CRCPress, Boca Raton, Fla., 1990, Vol. I), including meso-chlorin e₆monoethylenediamine (Mce₆), phytalocyanine, porphyrins and theirderivatives and analogs.

[0341] Suitably, the CD catalyst agent requires derivatives that providea “recognition site” and one or more “catalytic groups” on the CD agent(e.g. references; Ikeda, VanEtten, Hirai or Tabushi). Depending on theCD molecule used, the substrate to be catalyzed, and the reactionintended, the recognition site and catalytic groups can be providedthrough one or several derivatives, as needed. The recognition sitegenerally involves the hydrophobic cavity of the CD molecule, andprovides a means for specifically binding and/or orienting the substrateof interest with the CD catalytic agent.

[0342] The catalytic groups are generally organic and/or inorganicchemical residues, functional groups and ionic species that provide asuitable chemical environment for promoting the catalytic reaction. Thecatalytic groups can be any known chemical residue or species thatprovides the desired catalytic reaction, including carboxylates,imidazoles, histamines, hydroxyls, amines, amides, aldehydes, ketones,phosphates, sulfhydryls, halogens, amino adds, nucleic acids, chelators,and metals. Additional examples of suitable catalytic groups useful inthe instant invention can be found in the art of derivatizing CD's andderivatizing or “genetic engineering” of antibodies for use as enzymes.Other suitable references are; M. L. Bender, I. Tabushi E. Baldwin, P.G. Schultz (below).

[0343] In addition, an improved CD catalyst agent can be synthesizedwherein the recognition site and/or catalytic groups is provided oraugmented through the use of one or more suitable captured guests,described herein, that interact (e.g. binding, alignment and/or excimerformation), with the substrate being catalyzed. In this case, thecaptured guest is preferably coupled to the CD molecule by a suitablespacer group to allow interaction with the substrate, and can be anysuitable aliphatic, aromatic, or heterocyclic compound, including anysuitable inclusion compounds described herein.

[0344] Suitable CD catalyst agent reactions include hydrolysis (e.g. ofany suitable ester or amide containing substrates), oxidation,dephosphorylation, acid-base catalysis, formylation,dichloromethylation, carboxylation, rearrangement, substitution,allylation, and halogenation, among others. In any case, the catalyst CDagents can be prepared so that the catalyst CD product is inhibitory ortoxic to certain cells, microbes or parasites. The cyclodextrin catalystagents can also be coupled to a variety of substances, such asbiorecognition molecules, ligands, antigens, antibodies, nucleotides,nucleic acids, and liposomes, as well as to a variety of supportmaterials including magnetic particles for use in assays and chemicalprocesses.

[0345] An improved CD catalyst agent comprises the direct or indirectcoupling of any suitable antenna substance described herein, to the CDmolecule, for collection of light energy that is transferred to the CDcatalyst and thereby accelerates the desired reaction such as inphotodynamic therapy.

Preparation XV Amylose Polymer Carriers for Active Agents

[0346] The helical segments of amyloses, can be suitably polymerized,derivatized and/or capped to produce a carrier for drugs, nucleic acidsand other active agents wherein suitable functional and/or couplinggroups are included. Yet another composition includes the use of “selfassembly” substances coupled to the amylose.

[0347] Suitably, these amylose polymers have the necessary properties toform an inclusion complex with all or part of a nucleic acid, especiallythose with little or no net charge such as certain sense and antisenseODNs. Or, suitably, these amylose polymers have primary, secondary ortertiary amine groups to provide the necessary cationic charge forcomplexing with all or part of a suitable nucleic acid.

[0348] Preferred substances are soluble or colloidal polymers of helicalsegments of amyloses. Especially useful are helical amylose molecules ofmore than 5 and less than 120 glucose units, that favor formation ofrigid linear helixes.

[0349] In one preferred embodiment, a suitable amylose polymer oramylose segments are derivatized by various methods described herein forcyclodextrins, to provide cationic amine groups along the “edges” of theamylose chain. Then, a suitable anionic nucleic acid is mixed with thederivatized amylose to allow complexing to form between the cationicamylose and the anionic nucleic acid.

[0350] In another preferred embodiment, the amylose segments are firstderivatized before including a nucleic acid. For instance, some or allof the available hydroxyl groups are suitably thiolated by variousmethods described herein for thiolating cyclodextrins, to providesulfhydryl groups along the “edges” of the amylose chain.

[0351] Sulfhydryls are introduced through reaction of availablehydroxyls with a suitable epoxy compound. For instance, epichlorohydrinor a suitable diepoxy crosslinker previously described, is coupled to aCD or CD polymer wherein free epoxy groups are produced. Free epoxygroups are then reacted with sodium thiosulfate to give thiosulfateesters (e.g. Carlsson, supra). The thiosulfate esters are subsequentlyreduced to sulfhydryls with dithiothreitol.

[0352] Then, a nucleic acid is mixed with the derivatized amylose toallow inclusion complexes to form under mild reducing conditions. Afterthe nucleic acid is wholly or partially incorporated into the amylose,the complex is exposed to mild oxidation, which causes the sulfhydrylgroups to crosslink and entrap the nucleic acid in the amylose.

[0353] In another preferred embodiment, the amylose segments arederivatized by various methods described herein for cyclodextrins, toprovide amino groups along the “edges” of the amylose chain. Then, anucleic acid or other active agent is mixed with the derivatized amyloseto allow inclusion complexes to form. After the active agent is whollyor partially incorporated into the amylose, the complex is exposed toany suitable amino-specific crosslinking agent, which entraps the activeagent within the amylose.

[0354] Another preferred embodiment has incorporated cross-links thatcontain biocleavable linkages between the sulfhydryl or amino groups asdescribed previously.

[0355] Helical amylose polymers can be targeted by coupling them tobiorecognition molecules such as proteins, polypeptides, lipids,lipoproteins, nucleic adds, surfactants, virus coat proteins, andorganic molecules. They can include intermediate substances ofacrylamides (HPMA), PEG, nylons, polystyrenes, resins, metals andcelluloses, and their combinations.

Preparation XVI Micelle Polymer Carriers with Controlled Release

[0356] A micelle polymer carrier is a new invention defined herein as awater-soluble (or colloidal) micelle that has been suitably polymerizedso that it completely entraps a nucleic acid or other active agent. Theformation of micelles for carrying drugs, nucleic acids and other activeagents is well known. However, micelle carriers of the prior art sufferfrom uncontrolled loss of the drug due to diffusion. This inventionsolves that problem through cross-linking the micelle components tocompletely entrap the drug or other active agent until it is deliveredto the site of action.

[0357] For this invention, any suitable technology now used forpreparing drug-carrying micelles is applicable to the synthesis of thisinvention including disclosures of reagents for preparing liposomes andthose of Alkan-Onyuksel, H., Pharmaceutical Res. 11, 206-212 (1994). Adistinguishing property of this invention is that the micelle-formingcomponents must have suitable functional groups available on theirhydrophilic “heads” to permit cross-linking after the micelle has beenformed with a drug inside.

[0358] In one preferred embodiment, suitable micelles are formed thatcontain a drug. Then the head groups in the hydrophilic surface aresuitably cross-linked using various bifunctional cross-linking agents sothat the micelle cannot release the entrapped drug. Another preferredembodiment has incorporated cross-links that contain biocleavablelinkages as described previously. Also, this carrier can be suitablytargeted by coupling suitable biorecognition molecules to the surface.

Preparation XVII Amphiphilic Cyclodextrin Dimers, Trimers and Polymers

[0359] The purpose is to prepare a cure of water-soluble (or colloidal)amphiphilic cyclodextrin dimers, trimers and polymers with alkyl carbonchains attached. The cyclodextrins are cross-linked through hydroxylgroups using 1,4 butanediol diglycidyl ether (BDE) at low pH to favorreaction at the primary hydroxyl groups. Excess BDE molecules coupled atone end to the CD provide terminal oxirane groups that are subsequentlythiolated by reaction with thiosulfate and reduction. Alkyl carbonchains ate coupled to the CD derivatives using a “long chain epoxy” thatcouples to other available hydroxyl groups (CD88).

[0360] A. Cross-Linking with BDE.

[0361] Into 125 ml of hot water (70-80° C.) adjusted to pH 4.5-5 with0.05 ml 6 N HCl, was dissolved 2.84 gm of beta cyclodextrin (0.0025moles). To this solution 4.1 ml of BDE (about 0.0125 moles) was addedwith mixing and continued heating for about 2 hours.

[0362] B. Coupling with a Long Chain Epoxy.

[0363] The mixture was adjusted to pH>10 with KOH and 1.28 gm (about0.005 moles) of dodecyl/tetradecyl glycidyl ether (DTGE) was added andmixed vigorously. The solution was periodically mixed for about 1.5hours, heated for about 3 hours and then left at room temperature (rt)overnight The resulting solution was light yellow and turbid.

[0364] C. Coupling with Na Thiosulfate.

[0365] To the reheated mixture, 6 gm (about 0.025 moles) of sodiumthiosulfate was added and mixed. After about 1 hour, the pH was adjustedto 7 with KOH and the solution was heated for about 3.5 hours more.Excess DTGE was removed by chilling to solidify the DTGE and thesolution was decanted.

[0366] D. Dialysis.

[0367] The mixture was dialyzed against a continuous flow of distilledwater in 500 mwco tubing (Spectra/Por CE) for about 40 hours. Thesolution was concentrated by evaporation to 8 ml to give a clear, lightyellow solution.

[0368] E. Reduction with Dithiothreitol to Provide Thiol Groups.

[0369] To the mixture, 8 ml of water and 0.96 gm (about 0.0062 moles) ofdithiothreitol (DTT) was added, mixed and left overnight The turbidsolution was then dialyzed against a continuous flow of distilled waterin 500 mwco tubing (Spectra/Por CE) for about 40 hours. The solution wasconcentrated by evaporation to 3.7 ml to give a clear, yellow solution.Total yield based on dry weight was 2.276 gm of thiolated, amphiphiliccyclodextrin polymer.

[0370] F. Column Chromatography and Testing.

[0371] The mixture was fractionated on a Sephadex™ G15 column (2.5×47cm) in water. The fractions were tested for relative carbohydrate andthiol concentration.

[0372] Carbohydrate was tested for by combining: 0.012 ml of sample,0.01 ml of 1.5% 1-naphthol in methanol and finally 0.1 ml of 36N H₂SO₄to produce a color reaction. The absorbance was read at 620 nm andsample carbohydrate concentration was calculated by linear regressionusing values from a cyclodextrin standard curve.

[0373] Thiol groups were tested for by combining: 0.008 ml of sample and0.1 ml of 0.0125% 2,2′-dithio-bis(5-nitropyridine) (DTNP) in 62.5%isopropanol, pH 6 to produce a color reaction. The absorbance was readat 405 nm and sample thiol concentration was calculated by linearregression using values from a cysteine standard curve.

[0374] Fractions with corresponding peak concentrations of carbohydrateand thiol were pooled and concentrated by evaporation. The final volumewas 2.2 ml and the total yield based on dry weight was 1.144 gm.

[0375] The resulting mixture of thiolated amphiphilic CD dimers, trimersand polymers was highly water soluble and amorphous (glassy) when dried.

Preparation XVIII Cyclodextrin Dimers, Trimers and Polymers withBiocleavable Polypeptide Linkages

[0376] The purpose is to prepare cyclodextrin dimers, trimers andpolymers crosslinked through biocleavable polypeptide linkages. Thisreaction employs the hetero-bifunctional crosslinking agent,m-maliemidobenzoyl-N-hydroxysuccinimide ester (MBS) to crosslink anamino-containing cationic substance with the thiolated CD dimers,trimers and polymers of Preparation XVII (CD95). In this example, thebiocleavable linkage of leucine enkephalinamide, which contains thepeptide sequence: Tyr-Gly-Gly-Phe-Leu (Sigma) is coupled to thecyclodextrin dimers, trimers and polymers.

[0377] To about 0.1 ml of 50% dimethylformamide (DMF) and phosphatebuffered water, pH 6.5, containing about 0.0044 grams of leucineenkephalinamide, was added about 0.0036 gm of MBS dissolved in 0.1 mlDMF. This was mixed and allowed to react for about 30 minutes, whichproduced a clear solution. Then about 0.08 ml of water containing about0.028 gm of the thiolated mixture of CD dimers, trimers and polymers(Prep. XVII) was added and mixed. After about 1 hour, the solution wasclear but had turned brownish-orange, it was left overnight. The clear,brownish-orange mixture was exhaustively dialyzed first against 70%,then 35%, then 23% isopropanol, then finally distilled water in 2000mwco dialysis tubing (Sigma). During the dialysis, the clearbrownish-orange solution had turned very turbid with a precipitate,indicating a crosslinked product was produced.

Preparation XIX Cationic Cyclodextrin Dimers, Trimers and Polymers

[0378] The purpose is to prepare cyclodextrin dimers, trimers andpolymers with cationic groups attached. This reaction employs thehetero-bifunctional crosslinking agent,m-maliemido-benzoyl-N-hydroxysuccinimide ester (MBS) to crosslink anamino-containing cationic substance with the thiolated CD dimers,trimers and polymers of Preparation XVII (CD95).

[0379] To about 0.1 ml of 50% dimethylformamide (DMF) and phosphatebuffered water, pH 6.5, containing about 0.003 mmoles of neutralizedpolyethylenimine, mw about 800 (PEI800) was added about 0.0018 gm of MBSdissolved in 0.05 ml DMF. This was mixed and allowed to react for about20 minutes, which produced a turbid solution. Then about 0.08 ml ofwater containing about 0.028 gm of the thiolated mixture of CD dimers,trimers and polymers (Prep. XVII was added, mixed, and left overnight.

[0380] The mixture was exhaustively dialyzed first against 70%, then35%, then 23% isopropanol, then finally distilled water in 2000 mwcodialysis tubing (Sigma). The product was tested for binding to DNA(Promega DNA ladder) by first mixing 0.008 ml of 3-fold serially dilutedaqueous solutions of the cationic CD polymer preparation with equalvolumes containing about 1 microgram of DNA. These mixtures were thenrun on 2% agarose gel electrophoresis in Tris-borate EDTA buffer (withethidium bromide dye) at 60 volts, 36 milliamps for about 1 hour. Theresults were viewed and photographed over UV illumination. Resultsshowed that the DNA bands of the mixture diluted out 81-fold weresignificantly diminished compared to DNA alone. This indicates that thecationic mixture of amphiphilic CD dimers, trimers and polymers bound tothe DNA and inhibited migration through the gel.

Preparation XX Phosphoramidite-Adamantane

[0381] Using suitable synthesis methods based on those described andreferenced, phosphoramidite groups can be coupled to adamantane or othersuitable guests for use as CD linkers.

[0382] A new CD linker comprises phosphoramidite or other suitablenucleotide analog coupled to an adamantane dimer, adamantane trimer orsmall adamantane polymer (poly adamantane). This new linker issynthesized to allow incorporation into nucleic acids without adverselyaffecting their function and may also include suitable protective groupssuch as FMOC as needed. The resulting phosphoramidite-adamantane canthen be used in DNA synthesizing machines for production of guest-linkedoligonucleotides or ODNs or primers for PCR, with dimer or polyadamantane incorporated into their structure.

Preparation XXI Amino- and Thiol-Reactive Adamantane Dimers

[0383] A. Preparation of NHS-Adamantane and NHS-Adamantane Dimers.

[0384] In a suitable anhydrous solvent such as DMF, adamantanecarboxyateis combined with N-hydroxysuccinimide and an aromatic carbodiimide suchas N,N-dicyclohexylcarbodiimide, at approximately equimolar ratios andreacted at RT for 1-3 Hrs. The product, N-hydroxysuccinimide adamantane(NHS-Adamantane), is separated in the filtrate from precipitateddicyclohexylurea, collected by evaporation and purified by columnchromatography.

[0385] An adamantane dimer (or polymer) with an NHS ester group forcoupling is made by first coupling the adamantanes to a suitablesubstance with two (or more) available amino groups (suitablytemporarily protected if needed) and a carboxylate group. Some examplesare 3,5-diaminobenzoate, diaminopentanoic add (ornithine), lysine andcarboxylated low molecular weight polyethylenimine, among others. Forinstance 1-adamantane carbonyl chloride is first coupled to the aminogroups of 3,5-diaminobenzoate (or the temporarily protected ethyl esterif needed) in anhydrous conditions. The unprotected carboxylate group ofthe resulting 3,5-di-adamantane benzoate is then derivatized to an NHSester as described for NHS-Adamantane, above. The NHS ester-adamantanedimer can then be coupled to any suitable drug nucleic acid or otheractive agent with an available amino group.

[0386] B. Preparation of Thiol-Reactive Adamantane.

[0387] Alternatively, the 3,5-di-adamantane benzoate of A., above, isderivatized to a maleimido ester instead of an NHS ester, for sulfhydrylcoupling.

[0388] To prepare 2-bromo-N-acetamide adamantane, compounds 1-hydroxyadamantane or 1-adamantane methanol are derivatized based on theprocedure of B. Frisch, et al., Bioconj. Chem. 7,180-186 (1996).

[0389] To prepare maleimido adamantane, the compound 1-aminoadamantaneis coupled through the amino group tom-maleimidobenzoyl-N-hydroxysuccinimide ester, leaving the maleimidogroup available for coupling to a thiol group. To prepare iso-maleimidoadamantane, 1-aminoadamantane in suitable anhydrous solvent, is coupledthrough the amino group to maleic anhydride to give the maleamic acidderivative. The maleamic acid derivative is then dehydrated in suitableanhydrous solvent using an appropriate dehydrating agent such astrifluoroacetic anhydride to produce the iso-maleimido adamantaneavailable for coupling to an amino or thiol group.

[0390] While the invention has been described with reference to certainspecific embodiments, it is understood that changes may be made by oneskilled in the art and it would not thereby depart from the spirit andscope of the invention, which is limited only by the claims appendedhereto.

What is claimed is:
 1. A controlled release pharmaceutical compositioncomprising; a) cyclodextrin molecules selected from the group consistingof cyclodextrin derivatives, oxidized cyclodextrins, cyclodextrindimers, cyclodextrin trimers, and cyclodextrin polymers complexed with;b) an active agent selected from the group consisting of prodrugs,anticancer drugs, antineoplastic drugs, antifungal drugs, antibacterialdrugs, antiviral drugs, cardiac drugs, neurological drugs, alkaloids,antibiotics, bioactive peptides, steroids, steroid hormones, polypeptidehormones, interferons, interleukins, narcotics, prostaglandins, purines,pyrimidines, anti-protozoan drugs and anti-parasitic drugs wherein; c)said cyclodextrin molecules are covalently cross-linked through abiocleavable linkage to form a polymer that has entrapped the activeagent and wherein the cross-linking provides the function of controlledrelease.
 2. The composition of claim 1 wherein the biocleavable linkageis selected from the group consisting of disulfide linkages, protecteddisulfide linkages, ester bonds, aldehyde bonds, amide bonds,polypeptide linkages and hydrazone linkages.
 3. The composition of claim1 further comprising a biorecognition molecule coupled to thepharmaceutical composition.
 4. The composition of claim 1 wherein saidcyclodextrin dimers, cyclodextrin trimers, and cyclodextrin polymershave been derivatized to provide groups selected from the groupconsisting of dialdehydes, sulfobutylethers, sulfopropylethers,hydroxyethyls, hydroxypropyls, dihydroxy propyls, carboxylates andphosphates.
 5. The composition of claim 1 wherein the active agent isselected from the group consisting of ganciclovir, furosemide,indomethacin, camptothecins, cyclosporins, chlorpromazine, methotrexate,penicillin derivatives, anthracyclines, teramycins, tetracyclines,chlorotetracyclines, clomocyclines, butoconazole, ellipticines,guamecyclines, macrolides, filipins, fungichromins, nystatins,5′-fluorouracil, 5′-fluoro-2′-deoxyuridine, allopurinol and paclitaxe.6. The composition of claim 1 wherein said cyclodextrin molecules arecoupled to an intermediate coupling substance selected from the groupconsisting of serum albumins, glycoproteins, lipoproteins,polysaccharides, lipopolysaccharides, amino polysaccharides,polyacrylamides, lipids, glycolipids, N-(2-hydroxypropyl)methacrylamides, poly cyanoacrylates, polyethylene glycols, poly(D,L-lactic-coglycolic adds), dendrimers, poly(D,L-lactide)-block-methoxypolyethylene glycols and magnetic particles.7. A controlled release pharmaceutical composition comprising; a)cyclodextrin molecules selected from the group consisting ofcyclodextrin derivatives, oxidized cyclodextrins, cyclodextrin dimers,cyclodextrin trimers, and cyclodextrin polymers complexed with; b)nucleic acid, wherein; c) said cyclodextrin molecules are covalentlycross-linked through a biocleavable linkage to form a polymer that hasentrapped the active agent and wherein the cross-linking provides thefunction of controlled release.
 8. The composition of claim 7 whereinthe biocleavable linkage is selected from the group consisting ofdisulfide linkages, protected disulfide linkages, ester bonds, aldehydebonds, amide bonds, polypeptide linkages and hydrazone linkages.
 9. Thecomposition of claim 7 further comprising a biorecognition moleculecoupled to the pharmaceutical composition.
 10. The composition of claim7 wherein said cyclodextrin dimers, cyclodextrin trimers, andcyclodextrin polymers have been derivatized to provide groups selectedfrom the group consisting of dialdehydes, sulfobutylethers,sulfopropylethers, hydroxyethyls, hydroxypropyls, dihydroxy propyls,carboxylates and phosphates.
 11. The composition of claim 7 wherein thenucleic add is selected from the group consisting of DNA, RNA, sense andantisense oligonucleotides; sense and antisense oligodeoxynucleotides;sense and antisense oligonucleotides and oligodeoxynucleotidescontaining phosphodiesters, phosphorothioates, phosphorodithioates,phosphoroamidates, alkyl phosphotriesters, methylphosphonates,sulfamates, 3′-thioformacetals, methylene(methylimino)s,3′-N-carbamates, and morpholino carbamates; synthetic nucleic addpolymers, phosphoric acid ester nucleic acids and peptide nucleic acids.12. The composition of claim 7 wherein said cyclodextrin molecules arecoupled to an intermediate coupling substance selected from the groupconsisting of serum albumins, glycoproteins, lipoproteins,polysaccharides, lipopolysaccharides, amino polysaccharides,polyacrylamides, lipids, glycolipids, N-(2-hydroxypropyl)methacrylamides, poly cyanoacrylates, polyethylene glycols, poly(D,L-lactic-coglycolic acids), dendrimers, poly(D,L-lactide)-block-methoxypolyethylene glycols and magnetic particles.13. A controlled release pharmaceutical composition comprising; a)cyclodextrin molecules selected from the group consisting ofcyclodextrin derivatives, oxidized cyclodextrins, cyclodextrin dimers,cyclodextrin trimers, and cyclodextrin polymers complexed with; b)toxin, wherein; c) said cyclodextrin molecules are covalentlycross-linked through a biocleavable linkage to form a polymer that hasentrapped the active agent and wherein the cross-linking provides thefunction of controlled release.
 14. The composition of claim 13 whereinthe biocleavable linkage is selected from the group consisting ofdisulfide linkages, protected disulfide linkages, ester bonds, aldehydebonds, amide bonds, polypeptide linkages and hydrazone linkages.
 15. Thecomposition of claim 13 further comprising a biorecognition moleculecoupled to the pharmaceutical composition.
 16. The composition of claim13 wherein said cyclodextrin dimers, cyclodextrin trimers, andcyclodextrin polymers have been derivatized to provide groups selectedfrom the group consisting of dialdehydes, sulfobutylethers,sulfopropylethers, hydroxyethyls, hydroxypropyls, dihydroxy propyls,carboxylates and phosphates.
 17. The composition of claim 13 wherein theactive agent is selected from the group consisting of aflatoxins,ricins, bungarotoxins, irinotecan, pesticides, cevadines, desatrines,veratridine and cevine derivatives.
 18. The composition of claim 13wherein said cyclodextrin molecules are coupled to an intermediatecoupling substance selected from the group consisting of serum albuminsglycoproteins, lipoproteins, polysaccharides, lipopolysaccharides, aminopolysaccharides, polyacrylamides, lipids, glycolipids,N-(2-hydroxypropyl) methacrylamides, poly cyanoacrylates, polyethyleneglycols, poly (D,L-lactic-coglycolic acids), dendrimers, poly(D,L-lactide)-block-methoxypolyethylene glycols and magnetic particles.19. A pharmaceutical amylose composition comprising; a) amylose selectedfrom the group consisting of amylose segments, amylose derivatives,oxidized amylose and amylose polymers complexed with; b) an activeagent, wherein; c) said amylose is covalently cross-inked to form apolymer that has entrapped the active agent.
 20. A biocleavablecrosslinking agent comprising; a) a compound containing a biocleavablelinkage selected from the group consisting of polypeptide linkages andhydrazone linkages wherein; b) said compound has terminal reactivecoupling groups selected from the group consisting of N-succinimidyls,N-maleimidyls, p-nitrophenyl esters, iodoacetals, bromoacetals, oxiranesand imidoesters.
 21. A method for producing a cyclodextrinpharmaceutical composition comprising combining cyclodextrin moleculesselected from the group consisting of cyclodextrin derivatives,cyclodextrin dimers, cyclodextrin trimers, and cyclodextrin polymerswith; a) guest molecules coupled to a surface to form an inclusioncomplex between the cyclodextrin molecules and the guest molecules onthe surface, and; b) covalently cross-linking the cyclodextrin moleculesto form a polymer.
 22. A method for producing a cyclodextrinpharmaceutical composition using a solid support comprising coupling afirst cyclodextrin molecule selected from the group consisting ofcyclodextrin derivatives, cyclodextrin dimers, cyclodextrin trimers, andcyclodextrin polymers to a solid support through a cleavable couplingagent and; a) coupling in succession, additional cyclodextrin moleculesto the first cyclodextrin molecule that is coupled to the solid supportto form a polymer and; b) cleaving the first cyclodextrin molecule fromthe solid support.
 23. A pharmaceutical catalytic agent compositioncomprising; a) cyclodextrin molecules selected from the group consistingof oxidized cyclodextrins, cyclodextrin dimers, cyclodextrin trimers,and cyclodextrin polymers coupled with; b) a catalytic group selectedfrom the group consisting of carboxylates, imidazoles, histamines,hydroxyls, amines, amides, aldehydes, ketones, phosphates, sulfhydryls,halogens, amino acids, nucleic acids, chelators, and metals.